Glutathione S-transferases (GSTs) comprise a diverse superfamily of enzymes found in organisms from all kingdoms of life. GSTs are involved in diverse processes, notably small-molecule biosynthesis or detoxification, and are frequently also used in protein engineering studies or as biotechnology tools. Here, we report the high-resolution X-ray structure of Atu5508 from the pathogenic soil bacterium Agrobacterium tumefaciens (atGST1). Through use of comparative sequence and structural analysis of the GST superfamily, we identified local sequence and structural signatures, which allowed us to distinguish between different GST classes. This approach enables GST classification based on structure, without requiring additional biochemical or immunological data. Consequently, analysis of the atGST1 crystal structure suggests a new GST class, distinct from previously characterized GSTs, which would make it an attractive target for further biochemical studies.

Gye Won Han, Robert Schwarzenbacher, Rebecca Page, Lukasz Jaroszewski, Polat Abdubek, Eileen Ambing, Tanya Biorac, Jaume M. Canaves, Hsiu-Ju Chiu, Xiaoping Dai, Ashley M. Deacon, Michael DiDonato, Marc-André Elsliger, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Joanna Hale, Eric Hampton, Justin Haugen, Michael Hornsby, Heath E. Klock, Eric Koesema, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Inna Levin, Daniel McMullan, Timothy M. McPhillips, Mitchell D. Miller, Andrew Morse, Kin Moy, Edward Nigoghossian, Jie Ouyang, Jessica Paulsen, Kevin Quijano, Ron Reyes, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Frank von Delft, Xianhong Wang, Bill West, Aprilfawn White, Guenter Wolf, Qingping Xu, Olga Zagnitko, Keith O. Hodgson, John Wooley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, California The University of California, San Diego, La Jolla, California The Scripps Research Institute, La Jolla, California

Proteins: Structure, Function, and BioinformaticsVolume 61, Issue 2 p. 444-448 Structure Note Crystal structure of a putative modulator of DNA gyrase (pmbA) from Thermotoga maritima at 1.95 Å resolution reveals a new fold Chris Rife, Chris Rife The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorRobert Schwarzenbacher, Robert Schwarzenbacher The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorDaniel McMullan, Daniel McMullan The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPolat Abdubek, Polat Abdubek The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEileen Ambing, Eileen Ambing The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorHerbert Axelrod, Herbert Axelrod The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorTanya Biorac, Tanya Biorac The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJaume M. Canaves, Jaume M. Canaves The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorHsiu-Ju Chiu, Hsiu-Ju Chiu The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAshley M. Deacon, Ashley M. Deacon The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMichael DiDonato, Michael DiDonato The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMarc-André Elsliger, Marc-André Elsliger The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorAdam Godzik, Adam Godzik The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorCarina Grittini, Carina Grittini The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorSlawomir K. Grzechnik, Slawomir K. Grzechnik The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorJoanna Hale, Joanna Hale The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Hampton, Eric Hampton The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorGye Won Han, Gye Won Han The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJustin Haugen, Justin Haugen The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMichael Hornsby, Michael Hornsby The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorLukasz Jaroszewski, Lukasz Jaroszewski The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorHeath E. Klock, Heath E. Klock The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Koesema, Eric Koesema The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorAndreas Kreusch, Andreas Kreusch The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPeter Kuhn, Peter Kuhn The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorScott A. Lesley, Scott A. Lesley The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMitchell D. Miller, Mitchell D. Miller The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKin Moy, Kin Moy The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorEdward Nigoghossian, Edward Nigoghossian The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJessica Paulsen, Jessica Paulsen The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorKevin Quijano, Kevin Quijano The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRon Reyes, Ron Reyes The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorEric Sims, Eric Sims The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorGlen Spraggon, Glen Spraggon The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRaymond C. Stevens, Raymond C. Stevens The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorHenry van den Bedem, Henry van den Bedem The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJeff Velasquez, Jeff Velasquez The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJuli Vincent, Juli Vincent The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorAprilfawn White, Aprilfawn White The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorGuenter Wolf, Guenter Wolf The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorQingping Xu, Qingping Xu The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKeith O. Hodgson, Keith O. Hodgson The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJohn Wooley, John Wooley The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorIan A. Wilson, Corresponding Author Ian A. Wilson wilson@scripps.edu The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaThe Scripps Research Institute, BCC206, 10550 N. Torrey Pines Road, La Jolla, CA 92037===Search for more papers by this author Chris Rife, Chris Rife The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorRobert Schwarzenbacher, Robert Schwarzenbacher The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorDaniel McMullan, Daniel McMullan The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPolat Abdubek, Polat Abdubek The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEileen Ambing, Eileen Ambing The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorHerbert Axelrod, Herbert Axelrod The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorTanya Biorac, Tanya Biorac The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJaume M. Canaves, Jaume M. Canaves The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorHsiu-Ju Chiu, Hsiu-Ju Chiu The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAshley M. Deacon, Ashley M. Deacon The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMichael DiDonato, Michael DiDonato The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMarc-André Elsliger, Marc-André Elsliger The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorAdam Godzik, Adam Godzik The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorCarina Grittini, Carina Grittini The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorSlawomir K. Grzechnik, Slawomir K. Grzechnik The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorJoanna Hale, Joanna Hale The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Hampton, Eric Hampton The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorGye Won Han, Gye Won Han The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJustin Haugen, Justin Haugen The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMichael Hornsby, Michael Hornsby The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorLukasz Jaroszewski, Lukasz Jaroszewski The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorHeath E. Klock, Heath E. Klock The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Koesema, Eric Koesema The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorAndreas Kreusch, Andreas Kreusch The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPeter Kuhn, Peter Kuhn The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorScott A. Lesley, Scott A. Lesley The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMitchell D. Miller, Mitchell D. Miller The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKin Moy, Kin Moy The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorEdward Nigoghossian, Edward Nigoghossian The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJessica Paulsen, Jessica Paulsen The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorKevin Quijano, Kevin Quijano The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRon Reyes, Ron Reyes The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorEric Sims, Eric Sims The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorGlen Spraggon, Glen Spraggon The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRaymond C. Stevens, Raymond C. Stevens The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorHenry van den Bedem, Henry van den Bedem The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJeff Velasquez, Jeff Velasquez The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJuli Vincent, Juli Vincent The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorAprilfawn White, Aprilfawn White The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorGuenter Wolf, Guenter Wolf The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorQingping Xu, Qingping Xu The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKeith O. Hodgson, Keith O. Hodgson The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJohn Wooley, John Wooley The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorIan A. Wilson, Corresponding Author Ian A. Wilson wilson@scripps.edu The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaThe Scripps Research Institute, BCC206, 10550 N. Torrey Pines Road, La Jolla, CA 92037===Search for more papers by this author First published: 15 August 2005 https://doi.org/10.1002/prot.20468Citations: 9Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume61, Issue21 November 2005Pages 444-448 RelatedInformation

Proteins: Structure, Function, and BioinformaticsVolume 61, Issue 2 p. 449-453 Structure Note Crystal structure of the global regulatory protein CsrA from Pseudomonas putida at 2.05 Å resolution reveals a new fold Chris Rife, Chris Rife The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorRobert Schwarzenbacher, Robert Schwarzenbacher The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorDaniel McMullan, Daniel McMullan The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPolat Abdubek, Polat Abdubek The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEileen Ambing, Eileen Ambing The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorHerbert Axelrod, Herbert Axelrod The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorTanya Biorac, Tanya Biorac The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJaume M. Canaves, Jaume M. Canaves The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorHsiu-Ju Chiu, Hsiu-Ju Chiu The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAshley M. Deacon, Ashley M. Deacon The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMichael DiDonato, Michael DiDonato The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMarc-André Elsliger, Marc-André Elsliger The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorAdam Godzik, Adam Godzik The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorCarina Grittini, Carina Grittini The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorSlawomir K. Grzechnik, Slawomir K. Grzechnik The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorJoanna Hale, Joanna Hale The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Hampton, Eric Hampton The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorGye Won Han, Gye Won Han The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJustin Haugen, Justin Haugen The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMichael Hornsby, Michael Hornsby The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorLukasz Jaroszewski, Lukasz Jaroszewski The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorHeath E. Klock, Heath E. Klock The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Koesema, Eric Koesema The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorAndreas Kreusch, Andreas Kreusch The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPeter Kuhn, Peter Kuhn The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorScott A. Lesley, Scott A. Lesley The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMitchell D. Miller, Mitchell D. Miller The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKin Moy, Kin Moy The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorEdward Nigoghossian, Edward Nigoghossian The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJessica Paulsen, Jessica Paulsen The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorKevin Quijano, Kevin Quijano The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRon Reyes, Ron Reyes The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorEric Sims, Eric Sims The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorGlen Spraggon, Glen Spraggon The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRaymond C. Stevens, Raymond C. Stevens The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorHenry van den Bedem, Henry van den Bedem The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJeff Velasquez, Jeff Velasquez The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJuli Vincent, Juli Vincent The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorAprilfawn White, Aprilfawn White The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorGuenter Wolf, Guenter Wolf The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorQingping Xu, Qingping Xu The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKeith O. Hodgson, Keith O. Hodgson The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJohn Wooley, John Wooley The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorIan A. Wilson, Corresponding Author Ian A. Wilson wilson@scripps.edu The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaJCSG, The Scripps Research Institute, BCC206, 10550 North Torrey Pines Road, La Jolla, CA 92037===Search for more papers by this author Chris Rife, Chris Rife The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorRobert Schwarzenbacher, Robert Schwarzenbacher The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorDaniel McMullan, Daniel McMullan The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPolat Abdubek, Polat Abdubek The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEileen Ambing, Eileen Ambing The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorHerbert Axelrod, Herbert Axelrod The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorTanya Biorac, Tanya Biorac The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJaume M. Canaves, Jaume M. Canaves The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorHsiu-Ju Chiu, Hsiu-Ju Chiu The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAshley M. Deacon, Ashley M. Deacon The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMichael DiDonato, Michael DiDonato The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMarc-André Elsliger, Marc-André Elsliger The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorAdam Godzik, Adam Godzik The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorCarina Grittini, Carina Grittini The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorSlawomir K. Grzechnik, Slawomir K. Grzechnik The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorJoanna Hale, Joanna Hale The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Hampton, Eric Hampton The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorGye Won Han, Gye Won Han The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJustin Haugen, Justin Haugen The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMichael Hornsby, Michael Hornsby The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorLukasz Jaroszewski, Lukasz Jaroszewski The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorHeath E. Klock, Heath E. Klock The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Koesema, Eric Koesema The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorAndreas Kreusch, Andreas Kreusch The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPeter Kuhn, Peter Kuhn The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorScott A. Lesley, Scott A. Lesley The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorMitchell D. Miller, Mitchell D. Miller The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKin Moy, Kin Moy The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorEdward Nigoghossian, Edward Nigoghossian The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJessica Paulsen, Jessica Paulsen The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorKevin Quijano, Kevin Quijano The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRon Reyes, Ron Reyes The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorEric Sims, Eric Sims The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorGlen Spraggon, Glen Spraggon The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRaymond C. Stevens, Raymond C. Stevens The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorHenry van den Bedem, Henry van den Bedem The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJeff Velasquez, Jeff Velasquez The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJuli Vincent, Juli Vincent The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorAprilfawn White, Aprilfawn White The Joint Center for Structural Genomics The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorGuenter Wolf, Guenter Wolf The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorQingping Xu, Qingping Xu The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKeith O. Hodgson, Keith O. Hodgson The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJohn Wooley, John Wooley The Joint Center for Structural Genomics The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorIan A. Wilson, Corresponding Author Ian A. Wilson wilson@scripps.edu The Joint Center for Structural Genomics The Scripps Research Institute, La Jolla, CaliforniaJCSG, The Scripps Research Institute, BCC206, 10550 North Torrey Pines Road, La Jolla, CA 92037===Search for more papers by this author First published: 15 August 2005 https://doi.org/10.1002/prot.20502Citations: 43Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume61, Issue21 November 2005Pages 449-453 RelatedInformation

In order to extend the structural coverage of eukaryotic members of the Protein Family database (PFAM),1 we selected 400 open reading frames (ORFs) from available cDNA libraries of the Mouse genome. One of these, gi-13879369 belongs to the structurally and functionally uncharacterized protein family PF03674. This family is highly conserved, with hundreds of homologs in all kingdoms of life, and includes like BtrG from Bacillus circulans, which is part of the biosynthetic pathway for the antibiotic butirosin.2 Significant sequence homology is also found with the AIG2-like family (PF06094), which are plant proteins induced after bacterial infection.3 The 13879369 gene encodes a small protein with a molecular weight of 16,948 Da (residues 1–149) and a calculated isoelectric point of 5.1. Here, we report the crystal structure of 13879369, which was determined using the semi-automated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).4 A hypothetical protein from Mouse (gi: 13879369, IMAGE: 3501534, Swiss-Prot: Q923B0) was amplified by polymerase chain reaction (PCR) from a clone obtained from the IMAGE consortium using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends. The PCR product was cloned into plasmid pMH4, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a modified Terrific Broth using the Escherichia coli strain GeneHogs®. Lysozyme was added to the culture at the end of fermentation to a final concentration of 250 μg/mL. Bacteria were lysed by sonication after a freeze/thaw procedure in Lysis Buffer [50 mM Tris pH 7.9, 50 mM NaCl, 10 mM imidazole, 0.25 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)], and the cell debris was pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a nickel-chelating resin (Amersham Biosciences) pre-equilibrated with Lysis Buffer. The resin was washed with Wash Buffer [50 mM potassium phosphate pH 7.8, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP], and the target protein was eluted with Elution Buffer [20 mM Tris pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP]. The eluate was buffer-exchanged into Buffer Q [20 mM Tris pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl and applied to a RESOURCE Q column (Amersham Biosciences) pre-equilibrated with the same buffer. The target protein was eluted using a linear gradient of 50 to 500 mM NaCl in Buffer Q. The appropriate RESOURCE Q fractions were pooled and further purified using a Superdex 200 column (Amersham Biosciences) with elution in Crystallization Buffer [20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP]. The appropriate Superdex 200 fractions were pooled and concentrated for crystallization assays to 20 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method5 with standard JCSG crystallization protocols.4 The crystallization reagent contained 2.4 αM sodium formate, 0.1 αM sodium acetate (final pH 4.1). Twenty-five percent (v/v) glycerol (final concentration) was included as a cryoprotectant. The crystals were indexed in the hexagonal space group P65 (Table I). Highly redundant (∼60 fold) anomalous diffraction data were collected at the Advanced Light Source (ALS, Berkeley, USA) on beamline 8.3.1 at wavelength 1.743 Å (λ1) suitable for a Sulfur single-wavelength anomalous dispersion (SAD) experiment. The dataset was collected at 100K using an ADSC CCD detector. The data were integrated and scaled using HKL2000.8 Data statistics are summarized in Table I. The structure was determined with a highly redundant 1.90 Å Sulfur–SAD dataset using SHELX,9 SHARP,10 and ARP/wARP.11 Model completion and refinement were performed with XtalView12 and REFMAC5.6 Refinement statistics are summarized in Table I. The final model includes one monomer, residues 1–102, 106–149, two N-terminal histidines from the purification tag, three formic acid molecules (FMT), and 111 water molecules in the asymmetric unit. No electron density was observed for residues 103–105 and the rest of the expression and purification tag. Analysis of the stereochemical quality of the model was accomplished using AutoDepInputTool (http://deposit.pdb.org/adit/), MolProbity,13 SFcheck 4.0,6 and WHAT IF 5.0.14 Protein quaternary structure analysis used the PQS server (http://pqs.ebi.ac.uk/). Figures were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors have been deposited in the Protein Data Bank (PDB) and are accessible under the code 1vkb. The crystal structure of 13879369 [Fig. 1(a)] was determined to 1.90 Å resolution using the Sulfur–SAD method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes two monomers (residues 1–102 and 106–149), three formic acid molecules (FMT), and 111 water molecules in the asymmetric unit. The Matthews' coefficient (Vm)15 for 13879369 is 2.44 Å3/Da, and the estimated solvent content is 49.3%. The Ramachandran plot, produced by MolProbity,13 shows that 99.3% and 0.7% of the residues are in favored and allowed regions, respectively. Crystal structure of 13879369 from Mouse. (A) Ribbon diagram color-coded from N-terminus (blue) to C-terminus (red) showing the domain organization. Helices H1–H4 and β-strands β1–β7 are indicated. The partial disulfide bond between Cys35 and Cys62 is shown in ball-and-stick. (B) Diagram showing the secondary structure elements in 13879369 superimposed on its primary sequence. The α-helices, β-strands, β-bulges, γ-turns and disordered regions (dots) are indicated. The β-sheet strands are indicated by a red A and B. β-Hairpins are depicted as red loops. Protein 13879369 has approximate dimensions of 28 × 35 × 40 Å3 and is comprised of seven β-strands (β1–β7), three α-helices (H1, H3, H4), one 310 helix (H2) and extended loop regions [Fig. 1(a,b)]. The total β-strand, α-helical, and 310-helical contents are 34.5%, 9.5%, and 5.4%, respectively. Protein 13879369 folds into a five-stranded, antiparallel β-barrel (A) composed of strands β1, β2, and β5–β7. The β-barrel is flanked on one side by helices H1–H3, which pack against strands β1 and β5–β7. One of the open ends of the barrel interacts with β-sheet (B), composed of strands β3 and β4, which in turn packs against helix H4 and a long, C-terminal coil region. The β-barrel contains a disulfide bond between Cys35 and Cys62 adjacent to strands β2 and β5, respectively [Fig. 1(a,b)]. The electron density map shows alternative conformations for the cysteine side chains, indicating that the disulfide bond is only partially formed in the crystal, which may relate to the long exposure of the crystals to the intense X-ray beam during data collection.16 The structure contains a central, hydrophilic cavity gated by the strictly conserved residues Asn16 and Glu82 from the connecting regions following strand β1 and helix H3, and Tyr143 and Arg146 from the C-terminal, respectively, extended region [Fig. 2(a)]. The cavity is lined with Thr9 and four tyrosine residues (Tyr7, Tyr88, Tyr115, and Tyr143), and is solvent accessible through a narrow opening of approximately 2.5 Å in diameter adjacent to Asn16. The cavity is occupied by three water molecules and two FMT molecules: FMT1 is located at the cavity entrance within hydrogen-bonding distance of Arg146, whereas FMT2 is located deep inside the cavity next to Tyr7. The location of this cavity, its sequence conservation, and the presence of bound ligands points to its possible role as the active site of this protein. (A) Protein 13879369 in surface representation showing the putative active site cavity with neighboring residues in sticks. Two formic acid and three water molecules located inside the cavity are depicted in sticks and red balls, respectively. (B) Superposition of 13879369 (blue and red) and Ytfp (gray) in ribbon representation. Regions where backbones deviate by RMSD > 4 Å are colored red in 13879369. The two formates shown in red and yellow spheres indicate the position of the putative active site cavity. The crystallographic packing indicates that a monomer is the biologically relevant oligomeric form. A monomer is also consistent with results from analytical size exclusion chromatography and static light scattering. A structural similarity search, performed with the coordinates of the hypothetical protein using the DALI server,17 showed no structural similarity, indicating that 13879369 is a new fold. The recently solved structure of the hypothetical protein Ytfp from E. coli (PDB: 1xhs)18 shows significant similarity, with a root mean square deviation (RMSD) of 3.1 Å over 109 aligned residues with 22% sequence identity. Comparison with the shorter Ytfp structure (115 residues) reveals good overall agreement of both structures, but with three significant differences at the local structural level [Fig. 2(b)]. Ytfp contains an open β-barrel that shows no hydrogen bonding between strands β2 and β6. Ytfp contains an additional strand β8 that extends sheet B instead of the corresponding helix H4 that is found in the Mouse structure. In addition, the extended C-terminal coil region, which covers the cavity in the Mouse structure, is not present in Ytfp. Instead, Ytfp contains a large, positively charged crevice at this location, which supports another functional role for this region in the protein. Taken together, these structures suggest that the conserved hypothetical protein 13879369 from Mouse contains an internal active site and may function as an enzyme. This finding supports and adds to the current annotation of this protein and its protein family as a biosynthetic protein involved in cellular defense.2, 3 According to FFAS,19 the protein family including 13879369 has about 46 sequence homologs in eukaryotes, bacteria, and archaea. The Drosophila genome contains an uncharacterized entry YS11 (Q9WOY1) with 43% sequence identity. The closest sequence homologs in bacteria are the butirosin biosynthesis protein BtrG from Bacillus cereus, with 31% sequence identity, and the conserved hypothetical protein Ytfp from E. coli, with an overall sequence identity of 22%. Models for 13879369 homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=13879369. The 13879369 structure represents a conserved protein from Mouse whose structure has been determined by X-ray crystallography. The information reported here, in combination with further biochemical and biophysical studies, should yield valuable insight into the functional role of this protein in mammals. This work was supported by a NIH Protein Structure Initiative grant from the National Institute of General Medical Sciences (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL) and the Advanced Light Source (ALS). The SSRL is a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.

The TM1560 gene of Thermotoga maritima encodes a formiminotetrahydrofolate cyclodeaminase (FTCD_CD, EC 4.3.1.4), with a molecular weight of 22,581 Da (residues 1–202) and a calculated isoelectric point of 5.0.1 This enzyme is part of the folate metabolic pathway and catalyzes the cyclization of the formimino group of N5-formimidoyltetrahydrofolate, yielding N5,N10-methenyltetrahydrofolate and ammonia2 [Fig. 1(A)]. In eukaryotes, it is fused to glutamate formiminotransferase (FTCD_FT, EC 2.1.2.5), which catalyzes the previous step in the pathway (from tetrahydrofolate to N5-formiminotetrahydrofolate) to assemble the bifunctional enzyme, formiminotransferase-cyclodeaminase (FTCD; EC 2.1.2.5, EC 4.3.1.4).3 In Thermotoga maritima, FTCD_FT is encoded by a separate gene (TM0843). A genome context analysis reveals that close homologues of TM1560 form clusters with enzymes involved in folate metabolism in various bacterial genomes. For example, the cluster in Clostridium tetani and Thermoplasma volcani contains genes encoding glutamate formiminotransferase [Enzyme Commission (EC) 2.1.2.5) and formate-tetrahydrofolate ligase (EC 6.3.4.3). (A) Scheme for the reaction carried out by FTCD_CD (EC 4.3.1.4), which catalyzes the cyclization of the formimino group of N5-formimidoyltetrahydrofolate, yielding N5,N10-methenyltetrahydrofolate and ammonia. (B) Crystal structure of TM1560. Ribbon diagram of T. maritima FTCD_CD color-coded from N-terminus (blue) to C-terminus (red) showing the domain organization. Helices H1–H6 are indicated. (C) The TM1560 dimer view is shown along (above) and normal (below) to its two-fold axis. (D) Diagram showing the secondary structure elements in TM1560 superimposed on its primary sequence. The α-helices, 310-helices, β-bulges, and γ-turns are indicated. Disordered regions are depicted by a dashed line with the corresponding sequence in brackets. Here, we report the crystal structure of FTCD_CD determined using the semiautomated, high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).4 The crystal structure of FTCD_CD [Fig. 1(A)] was determined to 2.80 Å resolution using the single-wavelength anomalous dispersion (SAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes 2 protein monomers (residues 2–201 for chain A and residues 3–199 for chain B). The Matthews coefficient (Vm)7 for TM1560 is 2.84 Å3/Da and the estimated solvent content is 56.4%. The Ramachandran plot, produced by MolProbity,8 shows that 98.5% and 1.5% of the residues are in favored regions and additionally allowed regions, respectively. The final model of the TM1560 monomer consists of 6 α-helices (H1–H6) with three 310-helical segments (H3′, H5′, H6′), 8 β-turns, and 1 γ-turn [Fig. 1(B and D)]. The total α-helical and 310-helical content is 76.5% and 3.5%, respectively. The TM1560 monomer is composed of 6 α-helices arranged in an up-and-down helical bundle [Fig. 1(B)]. The crystallographic packing in the TM1560 structure suggests that a dimer is the biologically relevant oligomeric form with a buried surface area of 2301 Å2 per monomer. The dimer is formed through interactions of helices H1, H2, H5, and H6 [Fig. 1(C)] and contains 68% nonpolar atoms and 15 hydrogen bonds. A structural similarity search, performed with the coordinates of TM1560 using the DALI server,9 finds no structural homologues for the whole domain indicating that TM1560 is a new fold [Fig. 2(A)]. The closest structural homologue, the invertase inhibitor Nt-Cif from tobacco [Protein Data Bank (PDB) code: 1rj1],10 shows some similarity in the arrangement of helices H2–H5, but the helices in Nt-Cif are much shorter [Fig. 2(A)]. The root-mean-square deviation (RMSD) for this structural alignment is 3.2 Å over 109 aligned residues with 8% sequence identity. (A) Superposition of TM1560 (gray) and invertase inhibitor Nt-Cif from Tobacco (PDB code: 1rj1, magenta). (B) The putative active site pocket in surface representation. Residues are colored according to sequence conservation, where green is conserved and white is nonconserved. The manually docked ligand N5, N10-methenyltetrahydrofolate (MTHF) is shown in stick representation (yellow). (C) Same orientation as (B), but shown in ribbon representation, with conserved residues in the putative active site of TM1560 and the manually docked MTHF ligand in stick representation. Monomers 1 and 2 in the dimer are shown in blue and gray, respectively. The location of the TM1560 active site and its reaction mechanism are unknown. A map of conserved residues from an alignment of available FTCD_CD sequences reveals 2 identical clusters of conserved residues in the dimer interface. These clusters are located in a deep pocket (surface area: ∼800 Å2, volume: ∼1250 Å3) that is large enough to accommodate the product (N5, N10-methenyltetrahydrofolate, MTHF), which indicates a possible location of the FTCD_CD active site [Fig. 2(B and C)]. A similar placement of active sites in the dimer interface is also seen in glutamate formiminotransferase FTCD_FT.3 According to our modeling studies [Fig. 2(B and C)], the conserved residues can be divided into 2 groups according to their functional roles. The first group of residues (M41, F45, K49, K50, N131, N133, S136, and D137) from monomer 1 may be responsible for binding the p-aminobenzoyl portion of the ligand and catalysis. The second group (P20, G23, G24, G25, D80, F84, P109, N155, I158, and N159) from monomer 2, is located at the bottom of the groove, most likely binds and stabilizes the tetrahydropteridine ring of the substrate. This binding mode would put the strictly conserved aspartate, D137, close to the site where the deamination reaction occurs. Because aspartates or glutamates frequently serve as catalytic residues in deaminase reactions,11 we propose that D137 acts as the catalytic residue in FTCD_CD, and may extract a proton from the formiminogroup of the substrate to facilitate its deamination and subsequent cyclization. According to Fold and Function Assignment System (FFAS),12 the subfamily including TM1560 currently has 28 homologous sequences in all kingdoms of life. Models for TM1560 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM1560. The TM1560 structure reported here represents a formiminotetrahydrofolate cyclodeaminase from T. maritima, whose structure has been determined by X-ray crystallography using the SAD method. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the catalytic mechanism and functional determinants of this protein. Protein production and crystallization: Formiminotetrahydrofolate cyclodeaminase from T. maritima (TIGR: TM1560, Swiss-Prot: Q9X1P6) was amplified by polymerase chain reaction (PCR) from genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 250 μg/mL. Bacteria were lysed by sonication after a freeze-thaw procedure in Lysis Buffer [50 mM Tris, pH 7.9, 50 mM NaCl, 0.25 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)], and the cell debris was pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a nickel-resin (Amersham Biosciences) pre-equilibrated with Equilibration Buffer [50 mM potassium phosphate, pH 7.8, 300 mM NaCl, 10% (v/v) glycerol, 0.25 mM TCEP] containing 20 mM imidazole. The nickel-resin was washed with Equilibration Buffer containing 40 mM imidazole, and the protein was eluted with Elution Buffer [20 mM Tris, pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP]. Buffer exchange was performed to remove imidazole from the eluate, and the protein in Buffer Q [20 mM Tris, pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl was applied to a Resource Q column (Amersham Biosciences) pre-equilibrated with the same buffer. The protein was eluted using a linear gradient of 50 to 500 mM NaCl in Buffer Q. The appropriate fractions were pooled, buffer exchanged in Crystallization Buffer [20 mM Tris, pH 7.9, 150 mM NaCl, 0.25 mM TCEP] and concentrated to 12 mg/mL for crystallization by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method,13 with standard JCSG crystallization protocols.4 The crystallization reagent contained 5% polyethylene glycol 6000 (PEG-6000), 0.1 M citric acid at pH 5.0, and 35% glycerol (final concentration) was included as a cryoprotectant. The crystals were indexed in the orthorhombic space group P212121 (Table I). Data collection: Anomalous diffraction data were collected at the Advanced Light Source (ALS; Berkeley, CA) on beamline 8.2.2 at the wavelength corresponding to the peak (λ1) in a selenium SAD experiment (Table I). The data sets were collected at 100 K using a Quantum 315 charge-coupled device (CCD) detector. Data were integrated and reduced using MOSFLM14 and then scaled with the program SCALA from the CCP4 suite.5 Data statistics are summarized in Table I. Structure solution and refinement: The structure was determined by using the CCP4 suite5 and SOLVE/RESOLVE.15 Structure refinement was performed using REFMAC55 and Xfit.16 Refinement statistics are summarized in Table I. The final model includes 2 protein monomers (residues 2–201, 3–199) in the asymmetric unit. No electron density was observed for residues 1 and 202 in chain A, residues 1, 2, 200–202 in chain B, the side-chain atoms of R5, K19, K48, K50, E68, K89, Y91, K92, K170, and K192, and the rest of the expression and purification tags. Validation and deposition: Analysis of the stereochemical quality of the model was accomplished using the AutoDepInputTool (http://deposit.pdb.org/adit/), MolProbity,8 SFcheck 4.0,5 and WHATIF 5.0.17 Protein quaternary structure analysis used the PQS server (http://pqs.ebi.ac.uk/). Figure 1(B) was adapted from an analysis using PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/), and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of TM1560 have been deposited within the PDB and are accessible under the code 1o5h. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL) and the ALS. The SSRL is a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). The ALS is supported by the Director, Office of Science, Office of Basic Energy Sciences, Materials Sciences Division, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098 at Lawrence Berkeley National Laboratory.

Inna Levin, Mitchell D. Miller, Robert Schwarzenbacher, Daniel McMullan, Polat Abdubek, Eileen Ambing, Tanya Biorac, Jamison Cambell, Jaume M. Canaves, Hsiu-Ju Chiu, Ashley M. Deacon, Michael DiDonato, Marc-André Elsliger, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Joanna Hale, Eric Hampton, Gye Won Han, Justin Haugen, Michael Hornsby, Lukasz Jaroszewski, Cathy Karlak, Heath E. Klock, Eric Koesema, Andreas Kreusch, Peter Kuhn, Scott A. Lesley, Andrew Morse, Kin Moy, Edward Nigoghossian, Jie Ouyang, Rebecca Page, Kevin Quijano, Ron Reyes, Alyssa Robb, Eric Sims, Glen Spraggon, Raymond C. Stevens, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Xianhong Wang, Bill West, Guenter Wolf, Qingping Xu, Olga Zagnitko, Keith O. Hodgson, John Wooley, and Ian A. Wilson* Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California San Diego Supercomputer Center, La Jolla, California Genomics Institute of the Novartis Research Foundation, San Diego, California University of California, San Diego, La Jolla, California Scripps Research Institute, La Jolla, California

Proteins: Structure, Function, and BioinformaticsVolume 56, Issue 3 p. 629-633 Structure NoteFree Access Crystal structure of a putative NADPH-dependent oxidoreductase (GI: 18204011) from mouse at 2.10 Å resolution Inna Levin, Inna Levin The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorRobert Schwarzenbacher, Robert Schwarzenbacher The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorDaniel McMullan, Daniel McMullan The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPolat Abdubek, Polat Abdubek The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEileen Ambing, Eileen Ambing The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorTanya Biorac, Tanya Biorac The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJamison Cambell, Jamison Cambell The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJaume M. Canaves, Jaume M. Canaves The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorHsiu-Ju Chiu, Hsiu-Ju Chiu The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorXiaoping Dai, Xiaoping Dai The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorAshley M. Deacon, Ashley M. Deacon The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMichael DiDonato, Michael DiDonato The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorMarc-André Elsliger, Marc-André Elsliger The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorAdam Godzik, Adam Godzik The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorCarina Grittini, Carina Grittini The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorSlawomir K. Grzechnik, Slawomir K. Grzechnik The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorEric Hampton, Eric Hampton The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorLukasz Jaroszewski, Lukasz Jaroszewski The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorCathy Karlak, Cathy Karlak The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorHeath E. Klock, Heath E. Klock The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Koesema, Eric Koesema The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorAndreas Kreusch, Andreas Kreusch The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPeter Kuhn, Peter Kuhn The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorScott A. Lesley, Scott A. Lesley The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorTimothy M. McPhillips, Timothy M. McPhillips The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMitchell D. Miller, Mitchell D. Miller The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAndrew Morse, Andrew Morse The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorKin Moy, Kin Moy The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJie Ouyang, Jie Ouyang The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorRebecca Page, Rebecca Page The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorKevin Quijano, Kevin Quijano The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRon Reyes, Ron Reyes The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAlyssa Robb, Alyssa Robb The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Sims, Eric Sims The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorGlen Spraggon, Glen Spraggon The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorRaymond C. Stevens, Raymond C. Stevens The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorHenry van den Bedem, Henry van den Bedem The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJeff Velasquez, Jeff Velasquez The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJuli Vincent, Juli Vincent The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorFrank von Delft, Frank von Delft The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorXianhong Wang, Xianhong Wang The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorBill West, Bill West The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorGuenter Wolf, Guenter Wolf The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorQingping Xu, Qingping Xu The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKeith O. Hodgson, Keith O. Hodgson The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJohn Wooley, John Wooley The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, California The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorIan A. Wilson, Corresponding Author Ian A. Wilson [email protected] The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaJCSG, The Scripps Research Institute, BCC206, 10550 North Torrey Pines Road, La Jolla, CA 92037===Search for more papers by this author Inna Levin, Inna Levin The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorRobert Schwarzenbacher, Robert Schwarzenbacher The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorDaniel McMullan, Daniel McMullan The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPolat Abdubek, Polat Abdubek The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEileen Ambing, Eileen Ambing The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorTanya Biorac, Tanya Biorac The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJamison Cambell, Jamison Cambell The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJaume M. Canaves, Jaume M. Canaves The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorHsiu-Ju Chiu, Hsiu-Ju Chiu The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorXiaoping Dai, Xiaoping Dai The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorAshley M. Deacon, Ashley M. Deacon The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMichael DiDonato, Michael DiDonato The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorMarc-André Elsliger, Marc-André Elsliger The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorAdam Godzik, Adam Godzik The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorCarina Grittini, Carina Grittini The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorSlawomir K. Grzechnik, Slawomir K. Grzechnik The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorEric Hampton, Eric Hampton The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorLukasz Jaroszewski, Lukasz Jaroszewski The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorCathy Karlak, Cathy Karlak The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorHeath E. Klock, Heath E. Klock The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Koesema, Eric Koesema The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorAndreas Kreusch, Andreas Kreusch The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPeter Kuhn, Peter Kuhn The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorScott A. Lesley, Scott A. Lesley The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorTimothy M. McPhillips, Timothy M. McPhillips The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMitchell D. Miller, Mitchell D. Miller The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAndrew Morse, Andrew Morse The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorKin Moy, Kin Moy The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJie Ouyang, Jie Ouyang The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorRebecca Page, Rebecca Page The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorKevin Quijano, Kevin Quijano The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRon Reyes, Ron Reyes The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAlyssa Robb, Alyssa Robb The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Sims, Eric Sims The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorGlen Spraggon, Glen Spraggon The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorRaymond C. Stevens, Raymond C. Stevens The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorHenry van den Bedem, Henry van den Bedem The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJeff Velasquez, Jeff Velasquez The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJuli Vincent, Juli Vincent The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorFrank von Delft, Frank von Delft The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorXianhong Wang, Xianhong Wang The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorBill West, Bill West The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorGuenter Wolf, Guenter Wolf The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorQingping Xu, Qingping Xu The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKeith O. Hodgson, Keith O. Hodgson The Joint Center for Structural Genomics, Stanford University, Menlo Park, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJohn Wooley, John Wooley The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The San Diego Supercomputer Center, La Jolla, California The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorIan A. Wilson, Corresponding Author Ian A. Wilson [email protected] The Joint Center for Structural Genomics, Stanford University, Menlo Park, California The Scripps Research Institute, La Jolla, CaliforniaJCSG, The Scripps Research Institute, BCC206, 10550 North Torrey Pines Road, La Jolla, CA 92037===Search for more papers by this author First published: 20 May 2004 https://doi.org/10.1002/prot.20163Citations: 8AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL In order to extend the structural coverage of eukaryotic members in the Protein Family database (PFAM),1 we selected 400 open reading frames (ORF's) from the Mouse genome from available cDNA libraries. One of these, the mouse gene GI: 18204011 belongs to the structurally uncharacterized subfamily KOG11962 of zinc-containing dehydrogenases (PF00107),3 which has over 2000 homologues in all kingdoms of life. Thus, the 18204011 gene of mouse encodes a putative NADPH-dependent oxidoreductase, with a molecular weight of 37,921 Da (residues 1–351) and a calculated isoelectric point of 5.4. Here, we report the crystal structure of this putative oxidoreductase determined using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).4 The crystal structure of 18204011 [Fig. 1(A)] was determined to 2.10 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes one protein monomer (residues 1–253, 266–351) and 153 water molecules. No electron density was observed for residues 254–265. The Matthews' coefficient (Vm)5 for 18204011 is 2.60 Å3/Da and the estimated solvent content is 51.8%. The Ramachandran plot, produced by Procheck 3.4,6 shows that 93.7% of the residues are in the most favored regions and 6.3% are in additional allowed regions. Figure 1Open in figure viewerPowerPoint Crystal structure of 18204011. A: Stereo ribbon diagram of mouse 18204011 color-coded from N-terminus (blue) to C-terminus (red) showing the domain organization and location of the putative active site (arrow). Helices H1–H17, and β-strands (β1–β15) as well as β-sheets A, B, C and beginning (C253) and end (P266) of the disordered loop are indicated. B: Diagram showing the secondary structure elements in 18204011 superimposed on its primary sequence. The strands in each β-sheet are indicated by a red A, B, and C. β-hairpins are depicted as red loops. Disordered regions are depicted by a dashed line with the corresponding sequence in brackets. β-bulges are marked by β; γ-turns are marked by γ. Table I. Summary of Crystal Parameters, Data Collection, and Refinement Statistics for 18204011 (PDB: 1vj1) Space group P212121 Unit cell parameters a = 42.39 Å, b = 91.81 Å, c = 100.57 Å, α = β = γ = 90° Data collection λ1MADSe λ2MADSe λ3MADSe Wavelength (Å) 0.9795 0.9567 0.9793 Resolution range (Å) 50.28–2.10 100–50.35 50.39–2.60 Number of observations 135,157 99,705 90,496 Number of reflections 22,433 15,7.10 12,402 Completeness (%) 95.3 (74.9)a 98.5 (99.7)a 98.3 (99.6)a Mean I/σ(I) 10.9 (1.7)a 11.5 (2.8)a 9.8 (1.9)a Rsym on Ib 0.069 (0.431)a 0.074 (0.351)a 0.087 (0.495)a Sigma cutoff 0.0 0.0 0.0 Highest resolution shell (Å) 2.15–2.10 2.46–2.40 2.67–2.60 Model and refinement statistics Resolution range (Å) 50.28–2.10 Data set used in refinement λ1MADSe Number of reflections (total) 22,433 Cutoff criteria |F| > 0 Number of reflections (test) 1123 Rcrystc 0.180 Completeness (% total) 95.2 Rfreed 0.216 Stereochemical parameters Restraints (RMS observed) Bond length 0.016 Å Bond angle 1.47° Average isotropic B-value 19.6 Å2 ESU based on R valuee 0.20 Å Protein residues/atoms 341/2,567 Solvent molecules 153 a Highest resolution shell. b Rsym = Σ|Ii − Ii|/Σ|Ii| where Ii is the scaled intensity of the ith measurement, and Ii is the mean intensity for that reflection. c Rcryst = Σ| |Fobs| − |Fcalc| |/Σ|Fobs| where Fcalc and Fobs are the calculated and observed structure factor amplitudes, respectively. d Rfree = as for Rcryst, but for 5.0% of the total reflections chosen at random and omitted from refinement. e ESU = Estimated overall coordinate error.14, 18 The 18204011 monomer contains 15 β-strands (β1–β15), eight α-helices (H6, H7–H11, H13, H14), and nine 310-helices (H1–H5, H7′, H12, H13′, H14′) [Fig. 1(A, B)]. The total β-strand, α-helical, and 310-helical content is 29.0%, 22.6%, and 7.9%, respectively. The 18204011 structure is a member of the ζ-crystallin subfamily within the medium-chain dehydrogenase/reductase (MDR) superfamily.3 It comprises two distinct domains [Fig. 1(A, B)]: the catalytic domain (residues 1–132; 311–351) and the nucleotide-binding domain (residues 133–310). The catalytic domain contains nine β-strands arranged in two β-sheets (A and B) and six helices (H1–H5; H14). β-sheet A is antiparallel and composed of strands β1 and β2. β-sheet B is a highly twisted, seven-stranded (β3–β7, β14, β15) and forms a partly open, β-barrel-like structure with 67′41235 topology. Strands β3– β7 and β15 are antiparallel, whereas strand β14 is parallel to β15. Helices H5 and H13 to H14 form linker regions between the two domains. The nucleotide-binding domain folds into a three-layer αβα structure with a classical Rossman-fold. The domain comprises six β-strands (β8– β13) and eight helices (H6–H13), arranged as a six-stranded parallel β-sheet C, with 321456 topology, flanked by six α-helices. The domain contains an unusual nucleotide binding motive 162-GXXGXXG-168, with the GXGXXG being a much more common fingerprint of nucleotide binding. The present structure does not contain a bound nucleotide, which is most likely the reason for the observed disorder in one of the nucleotide-recognition loops (residues 254–265). In contrast to its annotation as a zinc-containing dehydrogenase, the 18204011 structure does not contain any zinc-binding residues that could form a metal center in its putative active site. The crystallographic packing in the 18204011 structure suggests that a monomer is the biologically-relevant form. A structural similarity search, performed with the coordinates of 18204011 using the DALI server,7 indicates structural similarity to a zinc-independent quinone oxidoreductase from E. coli (PDB: 1qor),8 with an RMSD of 3.4 Å over 326 aligned residues with 18% sequence identity [Fig. 2(A)]. Another structural homologue is enoyl thioester reductase (Etr1p) from Candida tropicalis (PDB: 1guf),9 where the RMSD is 3.4 Å over 305 aligned residues with 19% sequence identity. Figure 2Open in figure viewerPowerPoint A: Ribbon diagram of a superposition of 18204011 (mouse) and quinone oxidoreductase from E. coli (PDB: 1qor) grey. The structures were superimposed on their nucleotide-binding domains. The NADPH molecule bound to quinone oxidoreductase is shown in cpk mode. B: Close up view of the active site. The NADPH molecule and the sulfate bound to the active site of quinone oxidoreductase are shown in ball and stick. The active site tyrosine (Y52) as observed in quinone oxidoreductase from E. coli and its potential counterpart (Y64) in 18204011 (the Y64 side-chain has been modeled here due to disorder in the crystal structure) are shown in ball and stick. A detailed structural comparison with quinone oxidoreductase, which also does not contain zinc-coordinating residues, shows that the large cleft between the two domains in 18204011 could easily accommodate an NADPH molecule [Fig. 2(A, B)]. The relative disposition of the catalytic domains in the two structures shows that NADPH binding in quinone oxidoreductase is accompanied by a conformational change in the hinge region which moves the two domains closer to each other. This rigid body movement is observed in comparisons of the structures of the apo and NADPH-bound forms of quinone oxidoreductase.8 18204011 also contains positively-charged Lys192, as well as Tyr208, for the interaction with the phosphate group of NADPH. 18204011 also contains Gly 188, which is characteristic for proteins, exhibiting NADP rather than NAD specificity. The disordered loop between residues Cys253 and Pro266 comprises a conserved GxxS motif, which stabilizes both the adenine and nicotinamide moieties of the cofactor in the NADPH-bound form of quinone oxidoreductase. The analogy to quinone oxidoreductase indicates that 18204011 also binds NADPH and that its active site is also located in the groove next to helices H2, H5, and H6, which are adjacent to the nicotinate moiety of the bound NADPH molecule. The hydroxyl group of a catalytic tyrosine (or serine) acts as an electrophilic catalyst in the enzymatic reaction of quinone oxidoreductase and Etr1p.8, 9 18204011 contains Tyr64 [Fig. 2(B)], a potential counterpart for the catalytic tyrosine residue in quinone oxidoreductase (Tyr52) and enoyl thioester reductase (Tyr79). The Tyr64 side-chain is disordered in the 18204011 structure, which is most likely due to lack of any bound NADPH. Tyr64 appears to be conserved in the closest sequence homologues of 18204011 across all kingdoms of life, which suggests a redox mechanism related to that of quinone oxidoreductase.8 However, the reaction catalyzed by 18204011, awaits further biochemical studies and identification of its substrate and products. According to the Fold and Function Assignment System (FFAS),10 the subfamily including 18204011 has about one hundred homologous sequences in eukaryotic proteomes. Models for 18204011 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=18204011. The 18204011 structure reported here represents a putative NADPH-dependent oxidoreductase from mouse, whose structure has been determined by X-ray crystallography using the MAD method. The information reported here, in combination with further biochemical and biophysical studies will yield valuable insights into the functional determinants of this protein in mammals. Materials and Methods. Protein production and crystallization: A putative NADPH-dependent oxidoreductase from Mus musculus (GI: 18204011, IMAGE: 5068419, Swissprot: Q8VDQ1) was amplified by PCR from a clone obtained from the IMAGE consortium using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends. The PCR product was cloned into plasmid pMH4, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 250 μg/ml. Bacteria were lysed by sonication after a freeze-thaw procedure in Lysis Buffer (50 mM Tris, pH 7.9, 50 mM NaCl, 10 mM imidazole, 0.25 mM Tris[2-carboxyethyl]phosphine hydrochloride [TCEP]), and cell debris pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a nickel-resin (Amersham Biosciences) pre-equilibrated with Lysis Buffer. The nickel-resin was washed with Wash Buffer [50 mM potassium phosphate, pH 7.8, 300 mM NaCl, 40 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP], and the protein eluted with Elution Buffer [20 mM Tris, pH 7.9, 300 mM imidazole, 10% (v/v) glycerol, 0.25 mM TCEP]. Buffer exchange was performed to remove imidazole from the eluate, and the protein in Buffer A [20 mM Tris, pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP] containing 50 mM NaCl was applied to a Resource Q column (Amersham Biosciences) pre-equilibrated with Buffer A. The protein was eluted using a linear gradient of 50 to 500 mM NaCl in Buffer A. Appropriate fractions were further purified using a Superdex 200 column (Amersham Biosciences) with isocratic in crystallization buffer (20 mM Tris, pH 7.9, 150 mM NaCl, 0.25 mM TCEP). The protein was concentrated for crystallization assays to 18 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method11 with standard Joint Center for Structural Genomics crystallization protocols.4 The crystallization solution contained 15% polyethylene glycol (PEG) 4000, 0.2 M NH4-acetate, and 0.1 M Na-citrate (pH 5.1). The cryo-solution contained 15% ethylene glycol in addition. The crystals were indexed in the orthorhomic space group P212121 (Table I). Data collection. Anomalous diffraction data were collected at the Advanced Light Source (ALS, Berkeley, USA) on beamline 8.2.2 at wavelengths corresponding to inflection point (λ1), high energy remote (λ2), and peak (λ3) of a selenium MAD experiment, using the BLU-ICE12 data collection environment (Table I). The data sets were collected at 100 K using a Quantum 315 CCD detector. Data were integrated and reduced using MOSFLM13 and then scaled with the program SCALA from the CCP4 suite.14 Data statistics are summarized in Table I. Structure solution and refinement. The structure was determined by using the CCP4 suite14 and SOLVE/RESOLVE.15 Structure refinement was performed using REFMAC5,14 O,16 and Xfit.17 Refinement statistics are summarized in Table I. The final model includes one protein monomer (residues 1–253, 266–351), two histidine residues from the purification tag, and 153 water molecules in the asymmetric unit. No electron density was observed for residues 254–265, the side-chain atoms of D32, Y64, E115, E191, N212, R286, E297, K320, E347, or the rest of the expression and purification tag. Validation and deposition. Analysis of the stereochemical quality of the models was accomplished using PROCHECK 3.4 and SFcheck 4.0.7, 14 Figure 1(B) was adapted from an analysis using PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/) and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of 18204011 have been deposited within the PDB and are accessible under the code 1vj1. Acknowledgements This work was supported by NIH Protein Structure Initiative grant P50-GM 62411 from the National Institute of General Medical Sciences (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a National user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). REFERENCES 1 Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer EL. The Pfam protein families database. Nucleic Acids Res 2002; 30: 276– 280 2 Sun HW, Plapp BV. Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. 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J Appl Crystallogr 2002; 35: 278– 281. 12 McPhillips TM, McPhillips SE, Chiu HJ, Cohen AE, Deacon AM, Ellis PJ, Garman E, Gonzalez A, Sauter NK, Phizackerley RP, Soltis SM, Kuhn P. Blu-ice and the distributed control system: software for data acquisition and instrument control at macromolecular crystallography beamlines. J Synchrotron Radiat 2002; 9: 401– 406. 13 Leslie AGW. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsletter on Protein Crystallography 1992; 26. 14 Collaborative Computational Project Number 4. The CCP4 Suite: Programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 1994; 50: 760– 763. 15 Terwilliger TC, Berendzen J. Automated structure solution for MIR and MAD. Acta Crystallogr D Biol Crystallogr 1999; 55: 849– 861. 16 Jones TA, Zou J-Y, Cowan SW, Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr D Biol Crystallogr 1991; 47: 110– 119. 17 McRee, D.E. XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density. J Struct Biol 1999; 125: 156– 165. 18 Tickle IJ, Laskowski RA, Moss DS. Error estimates of protein structure coordinates and deviations from standard geometry by full-matrix refinement of gammaB- and betaB2-crystallin. Acta Crystallogr D Biol Crystallogr 1998; 54: 243– 252. Citing Literature Volume56, Issue315 August 2004Pages 629-633 FiguresReferencesRelatedInformation

The TM1112 gene of Thermotoga maritima encodes a conserved hypothetical protein with a molecular weight of 10,626 Da (residues 1–89) and a calculated isoelectric point of 5.5. Currently, no functional annotation has been made for this protein, but fold recognition methods, such as the Fold and Function Assignment System (FFAS),1 recognized significant sequence similarity to the family of cupins.2 Here, we report the crystal structure of TM1112 that was determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).3 The structure of TM1112 [Fig. 1(A)] was determined to 1.83 Å resolution by the molecular replacement (MR) method using the TM1112 NMR structure as the search model (PDB: 1lkn). Data collection, model, and refinement statistics are summarized in Table 1. The final model includes two protein monomers (residues 2–89), two unknown ligands (UNL), and 332 water molecules. The Matthews' coefficient (Vm) for TM1112 is 2.48 Å3/Da and the estimated solvent content is 50.0%. The Ramachandran plot, produced by Procheck 3.4,4 shows that 96.8% of the residues are in the most favored regions and 3.2% in additional allowed regions. Crystal structure of TM1112. A: Ribbon diagram of Thermotoga maritima TM1112 color coded from N-terminus (blue) to C-terminus (red) showing the domain organization viewed along (left) and normal (right) to the barrel axis. Helices (H1, H2), β-sheets (A and A′), and β-strands (β1–β7) are indicated. B: Diagram showing the secondary structure elements in TM1112 superimposed on its primary sequence. The β-sheets are indicated by a red A or A′ and the β-hairpin is depicted as red loops. Residues adjacent to the the unknown ligand (UNL) molecule are marked with red dots (also see Fig. 2). A: The proposed active site of TM1112 is depicted with the unknown ligand molecule (UNL) bound to Lys84 and its coordinating residues (Trp24, Trp33, Glu39, Cys41, Tyr35, and Trp76) in ball and stick. B: Close up view of the active site with a 2Fo–Fc map around Lys84, the covalently-bound UNL and Cys41 contoured at 1σ (marine blue). The atoms are indicated as follows: carbon (grey), oxygen (red), nitrogen (blue), sulfur (yellow), and UNL (pink). Potential covalent bonds for the UNL ligand are represented as dashed pink lines, but until ligand identification, these are quite speculative. The TM1112 monomer is composed of seven β-strands (β1–β7), one α-helix (H1), and one short 310-helix (H2). The total β-strand content is 59.1%. The TM1112 structure is characterized by an antiparallel β-sheet that forms a jelly roll β-sandwich with a topology that is reminiscent of the cupin barrel fold2 [Fig. 1(A)]. The seven-stranded β-sheet (β1–β7) can be viewed as composed of two connected β-sheets, A with 16472 topology and A′ with 3745 topology, fused together via two strongly bent β-strands β4 and β7 [Fig. 1(A)]. Because of this variation, the Structural Classification of Proteins database (SCOP)5 classified TM1112 as a new subfamily of RmlC-like cupins. The root-mean-square deviation (RMSD) between the crystal structure and the averaged NMR structure (PDB: 1lkn) of TM1112 is 1.3 Å over 88 aligned residues. Both structures indicate that a monomer is the biologically-relevant form of TM1112. An alignment of the TM1112 sequence with homologous-cupin-like sequences, derived from a FFAS1 search, identifies a cluster of strictly conserved residues (Trp24, Trp33, Glu39, Cys41, Tyr35, Trp76, and Lys84) located in the center of the β-barrel [Fig. 2(A)]. The conservation of these side-chains within a groove in the center of the β-barrel indicates a proposed location for the TM1112 active site. SigmaA-weighted OMIT maps show additional compact density contiguous with the side-chain amino group of Lys84 [Fig. 2(B)]. Connecting density also suggests a hydrogen bond (distance 2.75 Å) between Lys84 and the adjacent sulfhydryl group of Cys41. Despite extensive model building and database searching, the density could not be unambiguously interpreted and was, therefore, modeled as an unknown ligand (UNL) consisting of five atoms covalently bound to Lys84 which suggests a catalytic role for Lys84 and Cys41. The apparent covalent nature of this adduct suggests either a post-translational modification or interaction with an unknown substrate. However, we were unable to identify a similar active site configuration in the PDB which indicates that TM1112 represents a functionally novel enzyme from the cupin family. Clearly, further work is needed to define the enzymatic activity and mechanism of these cupins. A structural similarity search, performed with the coordinates of TM1112 using the DALI server,6 indicated that the closest structural homologue is quercetin 2,3-dioxygenase, an RmlC-like cupin from Aspergillus japonicus (PDB: 1 juh),7 with an RMSD of 2.4 Å over 82 aligned residues with 12% sequence identity. Another structural homologue is the N-terminal domain of the Catabolite Gene Activator Protein (CAP) from Escherichia coli (PDB: 2cgp),8 where the RMSD is 2.7 Å over 79 aligned residues with 11% sequence identity. According to FFAS,1 TM1112 has four distant homologues in the Thermotoga maritima proteome: TM1010 with 10% sequence identity, TM1287 (14%), TM1459 (10%), and TM0656 (14%). Sequence similarity searches with the TM1112 sequence against the non-redundant protein sequence database (NCBI) revealed more than one hundred homologues in prokaryotes and eukaryotes, all of which are designated as conserved hypothetical proteins. This new cupin sub-family comprises single-domain proteins like TM1112, as well as multi-domain proteins. Models for TM1112 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM1112. The crystal structure reported here represents a novel enzyme from the cupin family that was determined by MR using the TM1112 NMR structure as a template. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the functional determinants of this protein and the thermostability of these organisms. TM1112 (TIGR: TM1112; Swissprot: Q9X0J6) was amplified by polymerase chain reaction (PCR) from Thermotoga maritima strain MSB8 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of TM1112. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a modified Terrific Broth [24 g/liter yeast extract, 12 g/liter tryptone, 1% (v/v) glycerol, 50 mM 3-(N-Morpholino)propanesulfonic acid (MOPS), pH 7.6] using the E. coli strain GeneHogs® (Invitrogen). Lysozyme was added to the culture at the end of fermentation to a final concentration of 1 mg/ml. Bacteria were lysed by sonication after a freeze-thaw procedure in Lysis Buffer [50 mM Tris, pH 7.9, 50 mM NaCl, 1 mM MgCl2, 5 mM 2-Mercaptoethanol, 3 mM DL-methionine, 2.5 U/ml Benzonase® (Sigma)], and cell debris pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a nickel-resin (Amersham Biosciences) pre-equilibrated with Equilibration Buffer (50 mM potassium phosphate, pH 7.8, 0.25 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), 10% (v/v) glycerol, 400 mM NaCl, 100 mM KCl, 20 mM imidazole, 3 mM DL-methionine). The nickel-resin was washed with Equilibration Buffer, and the protein eluted with Elution Buffer (20 mM Tris, pH 7.9, 10% (v/v) glycerol, 0.25 mM TCEP, 200 mM imidazole, 3 mM DL-methionine). The eluate was buffer exchanged into Crystallization Buffer (20 mM Tris, pH 7.9, 150 mM NaCl, 0.25 mM TCEP) and concentrated for crystallization assays to 19 mg/ml by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method9 using standard JCSG crystallization protocols.3 Crystals grew in Hampton Crystal Screen Cryo #31 [25.5% polyethylene glycol (PEG) 4000, 15% glycerol, and 0.17 M (NH4)2SO4]. The crystals were indexed in the monoclinic space group P21 (Table I). Native diffraction data were collected on beamline 9-1 at the Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, USA) using the BLU-ICE10 data collection environment (Table I). The dataset was collected at 100K using a Quantum 315 CCD detector. Data were integrated and reduced using Mosflm11 and then scaled with the program SCALA from the CCP4 suite.12 Data statistics are summarized in Table I. The structure was determined by molecular replacement using the program MOLREP from the CCP4 suite.12 The ten models from the NMR structures of TM1112 (PDB: 1lkn), solved by the Northeast Structural Genomics Consortium,13 were used as search models. The correct solution could only be obtained with model number 9 and gave an Rfree = 0.48 and Rcryst = 0.46 after initial rigid body and restrained refinement in REFMACS.12 Structure refinement was performed using TLS refinement in REFMAC5,12 O,14 and Xfit.15 Refinement statistics are summarized in Table I. The final model includes two protein monomers (residues 2–89), two unknown ligands (UNL), and 332 water molecules in the asymmetric unit. No electron density was observed for the expression or purification tag. Analysis of the stereochemical quality of the models was accomplished using Procheck 3.4,4 SFcheck 4.0,12 and WHAT IF 5.0.16 Figure 1(B) was adapted from an analysis using PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/) and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates of the final model and experimental structure factors of TM1112 have been deposited with the PDB and are accessible under the code 1o5u. This work was supported by NIH Protein Structure Initiative grant P50-GM 62411 from the National Institute of General Medical Sciences (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences).

The TM1459 gene of Thermotoga maritima encodes a conserved hypothetical protein with a molecular weight of 12,977 Da (residues 1–114) and a calculated isoelectric point of 5.6. Currently, no functional annotation has been made for this protein, made fold recognition methods such as Fold and Function Assignment System (FFAS)1 have recognized significant sequence similarity to the family of cupins.2 Here, we report the crystal structure of TM1459 with an endogenous manganese ligand that was determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).3 The structure of TM1459 [Fig. 1(a)] was determined to 1.65 Å resolution using the molecular replacement (MR) method using a search model constructed from oxalate oxidase (PDB: 1FI2),4 despite a very low sequence identity (18%). Data collection, modeling and refinement statistics are summarized in Table I. The final model includes two protein molecules (residues 1–114), 2 manganese ions, and 252 water molecules. The Matthews' coefficient (Vm) for TM1459 is 2.71 Å3/Da, and the estimated solvent content is 54.2%. The Ramachandran plot, produced by Procheck 3.4,5 shows that 92.7% of the residues are in the most favored regions, and 7.3% are in additional allowed regions. Crystal structure of TM1459 shown as (a) ribbon diagram of Thermotoga maritima TM1459 color coded from N-terminus (blue) to C-terminus (red) showing the domain organization and the location of the manganese ion (purple sphere). The 310-helices (H1, H2), β-strands (β1–β11 and β-sheets A and B are indicated; and (b) ribbon diagram of TM1459 dimer. β-strands β1 and β9 of the crossover domain-swap interaction are indicated and are labeled β1′ and β9′ in the opposing subunit. The final model of the TM1459 monomer is composed of 11 β-strands (β1–β11) and two short 310-helices (H1, H2). The total β-strand content is 43.6%. The TM1459 structure is characterized by two antiparallel β-sheets (A and B) that form a jellyroll β-sandwich with a topology that is characteristic of the cupin-barrel fold3, 4 [Fig. 1(a)]. The seven-stranded β-sheet A (β1′, β2–β4, β6, β9, β11) has a 2347561 topology, where β-strand β1′ is contributed from the neighboring subunit of the dimer by domain-swapping (Fig. 1). The four-stranded β-sheet B (β5, β7, β8, β10) has 1423 topology [Fig. 1(a)]. Each of the 11 β-strands runs approximately perpendicular to the barrel axis. TM1459 forms a dimer linked by a pair of crossovers between the adjacent edge β-strands β1 and β9 from different subunits in the dimer [Fig. 1(b)]. The dimer interface corresponds to interactions between the two A β-sheets with a buried surface area of 863 Å2 per monomer.6 Each TM1459 domain has a metal-binding site in the mouth of the β-barrel [Fig. 2(b)]. The metal ion has octahedral coordination in which four ligands are contributed by conserved histidine side chains that define a new sub-family of cupins. The metal coordination is different from the His, His, Glu, His metal-coordination typically found in cupins and is the second example of a metal coordinated by four histidine residues similar to that observed for manganese in the photosynthetic reaction center of Rhodobacter sphaeroides (PDB: 1YST).7 The metal-binding residues are His52, His54, His58, and His92 with metal-to-atom distances of 2.19, 2.19, 2.25 and 2.13 Å, respectively. The remaining coordination sites are occupied by two water molecules (W1, W2) at distances of 2.06 and 2.24 Å [Fig. 2(b)]. The metal has been assigned as manganese because it gave the best refined B-factor agreement with surrounding atoms (tested for a series of metals) and also based on the identity (Mn) and similar octahedral coordination of the metal in the structural homologue oxalate oxidase.4 Sigma-A-weighted OMIT maps show density continuous with the sulfhydryl group of Cys106, which in subunit A extends as a continuous tube past the Mn-coordinated water molecules. Although the density could not be identified and was modeled as an unknown ligand (UNL) H-bonded to Cys106 [Fig. 2(b)], it does suggest a catalytic role for Cys106. (a) Diagram showing the secondary structure elements in TM1459 superimposed on its primary sequence. β-Hairpins are depicted as red loops. Residues coordinating the metal ion are marked with blue dots. (b) The active site of TM1459 showing the proposed manganese ion (Mn), its coordinating residues (His52, His54, His58, His92, and two waters) and Cys106, adjacent to the unidentified density modeled as UNL, is depicted in ball and stick. The atoms are indicated as follows: carbon (grey), oxygen (red), nitrogen (blue), sulfur (orange) and manganese (purple). Metal-ligand bonds are represented as dashed yellow lines. A structural similarity search, performed with the coordinates of TM1459 using the DALI server,8 indicated that the closest structural homologue is oxalate oxidase (germin) from Hordeum vulgare (PDB: 1FI2), which was used here as a MR search model.4 The RMSD between TM1459 and oxalate oxidase is 1.4 Å over 74 aligned residues with 20% sequence identity. Another structural homologue is TM1287, a putative oxalate decarboxylase whose structure was determined recently (PDB: 1O4T).9 The RMSD between TM1459 and TM1287 is 1.7 Å over 80 aligned residues with 18% sequence identity. According to FFAS,1 TM1459 has at least five distant homologues in the Thermotoga proteome: TM1287 (18% sequence identity), TM1010 (16%), TM0656 (13%), TM1112 (10%), and TM0736 (8%). Sequence similarity searches with the TM1459 sequence against the non-redundant protein sequence database revealed more than 1000 homologues in the three kingdoms of life, with about 100 homologues from this new sub-family that contain metals coordinated by four histidines. This subfamily comprises single-domain proteins like TM1459, as well as multi-domain proteins like the family of mannose phosphorylases. Models for TM1459 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=TM1459. The crystal structure reported here is the first representation of a novel subfamily of cupins that contains a metal site coordinated by four histidines. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the functional determinants of this protein family and the thermostability of these organisms. TM1459 (TIGR: TM1459; Swissprot:Q9X1H0) was amplified by PCR from Thermotoga maritima strain MSB8 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of TM1459. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag (MGSDKIHHHHHH) at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a modified Terrific Broth [24 g/L yeast extract, 12 g/L tryptone, 1% (v/v) glycerol, 50 mM 3-(N-Morpholino) propanesulfonic acid (MOPS) pH 7.6] using the Escherichia coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 1 mg/mL. Bacteria were lysed by sonication after a freeze–thaw procedure in Lysis Buffer [50 mM Tris pH 7.9, 50 mM NaCl, 1 mM MgCl2, 0.25 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP)] and the cell debris pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a metal chelate affinity resin (Amersham Biosciences) and equilibrated with Equilibration Buffer [50 mM potassium phosphate pH 7.8, 0.25 mM TCEP, 10% (v/v) glycerol, 300 mM NaCl] containing 20 mM imidazole. The Ni-resin was washed with Equilibration Buffer containing 40 mM imidazole, and the protein was eluted with Elution crystallization buffer [20 mM Tris pH 7.9, 10% (v/v) glycerol, 0.25 mM TCEP, 300 mM imidazole]. The eluate was buffer exchanged into crystallization buffer (20 mM Tris pH 7.9, 150 mM NaCl, 0.25 mM TCEP) and concentrated to ≈10 mg/mL for crystallization trials by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method10 with standard JCSG crystallization protocols.3 The crystallization solution contained 50% PEG 200, 0.2M NaCl, and 0.1M Na-phosphate-citrate (pH 4.2) (final pH 5.2). The crystals were indexed in the trigonal space group P32 (Table I). Native diffraction data were collected at Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, USA) on beamline 11-1 using the BLU-ICE11 data collection environment (Table I). The data set was collected at 100K using a Quantum 315 charge-coupled device (CCD) detector. Data were integrated and reduced using Mosflm12 and then scaled with the program SCALA from the CCP4 suite.13 The crystal suffered from partial twinning (twinning fraction 0.35) and data were detwinned with program XPREP. Data statistics are summarized in Table I. The structure was determined by molecular replacement using program MOLREP from the CCP4 suite.13 A homology model based on the FFAS1 alignment between TM1459 and oxalate oxidase (PDB: 1FI2), with a sequence identity of only 18%, was constructed with the modeling program Whatif14 and used as a search model. Structure refinement was performed using REFMAC5,13 O15 and Xfit.16 Refinement statistics are summarized in Table I. The final model includes a protein dimer (residues 1–114), two manganese ions, and 252 water molecules in the asymmetric unit. No electron density was observed for the expression or purification tag. Analysis of the stereochemical quality of the models was accomplished using Procheck 3.4 and SFcheck 4.0.5, 13 Protein quarternary structure analyis and buried surface area were taken from the PQS server (http://pqs.ebi.ac.uk/). Atomic coordinates of the final model and experimental structure factors of TM1459 have been deposited with the PDB and are accessible under the code 1o5n. This work was supported by NIH Protein Structure Initiative grant P50-GM 62411 from the National Institute of General Medical Sciences (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences).

The AF0591 gene of Archaeoglobus fulgidus (DSM4304; gi-number: 2650039) encodes a conserved hypothetical protein, with a molecular weight of 15,335 Da (residues 1–135) and a calculated isoelectric point of 8.5. In order to extend the structural coverage of the Protein Family database (PFAM),1 we selected 331 bacterial representatives with homologues in the mouse genome from the American Type Culture Association (ATCC) proteome database (http://www.ebi.ac.uk/proteome/proteomesource.html). One of these representatives, AF0591, contains a PIN (PilT N-terminus) domain(PF01850),2 which has over 300 homologues in all kingdoms of life. The PIN domain in the PilT N-terminus is a compact domain of about 100 amino acids which polymerizes to a pilus fiber.3 In addition, the PIN domain appears to have a role in signaling given its presence in some bacterial plasmid stability proteins and in Dis3 from yeast that is implicated in mitotic control.4 Here, we report the crystal structure of AF0591 determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).5 The structure of AF0591 [Fig. 1(A)] was determined to 1.90-Å resolution using the multiwavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes 1 protein molecule (residues 10–134) and 115 water molecules. No electron density was observed for residues 1–9 and 135. The Matthews coefficient (Vm)6 for AF0591 is 3.04 Å3/Da, and the estimated solvent content is 59.2%. The Ramachandran plot, produced by Procheck 3.4,7 shows that 98% of the residues are in the most favored regions, and 2% are in additional allowed regions. Crystal structure of AF0591. (A) Ribbon diagram of Archaeoglobus fulgidus AF0591 color coded from N-terminus (blue) to C-terminus (red) showing the domain organization. The disulfide-bond between Cys13 and Cys100 is shown in ball and stick (orange). α-Helices H1–H6, and β-strands (β1– β5) in β-sheet A are indicated. (B) Diagram showing the secondary structure elements in AF0591 superimposed on its primary sequence. The disulfide-bond (Cys13–Cys100) is indicated, and the disordered regions are depicted by a dashed line with the corresponding sequence in brackets. Strands are labeled according to their β-sheet (A). The location of γ-turns are also indicated. Mse refers to seleno-methionine. The final model of the AF0591 monomer is composed of 5 β-strands (β1–β5) and 6 α-helices (H1–H6) [Fig. 1(A and B)]. The total β-strand and α-helical content is 16.0% and 51.2%, respectively. AF0591 folds into a α/β/α domain with a central, 5-stranded, parallel β-sheet, comprised of β-strands β1–β5, with 32145 topology and extended, 2-helical hairpin connections between β-strands β1 and β2, and β2 and β3. The β-sheet is twisted and surrounded by 6 α-helices (H1–H6). The structure contains a disulfide bond between Cys13 and Cys100 [Fig. 1(A and B)]. A structural similarity search, performed with the coordinates of AF0591 using the DALI server,8 indicates distant structural similarity for the central β-sheet of AF0591 to the catalytic domain (53EXOc) of 5′-3′ exonuclease from Bacteriophage T5 [Protein Data Bank (PDB) code: 1EXN].9 The root-mean-square deviation (RMSD) is 3.2 Å over 101 aligned residues with 14% sequence identity. Because the structural resemblance is limited only to the central β-sheet and α-helices H2, H5, and H6 [Fig. 2(B)], the structure of AF0591 can be considered a new fold. (A) Ribbon diagram of the AF0591 dimer. (B) Ribbon diagram of a superposition of AF0591 (rainbow) and the catalytic domain (residues 1–174) of 5′-3′ exonuclease from Bacteriophage T5 (gray). α-Helices and β-strands are indicated for AF0591. (C) Close-up view of the active site. Active site residues as observed in T5 5′-3′ exonuclease (labels shown in brackets) and their counterparts in AF0591 are shown in ball and stick. Colors are the same as in (B). The side-chain of E108 in AF0591 was disordered and, therefore, not modeled. For the T5 5′-3′ exonuclease, the active site was reported9 to be formed by a cluster of 7 conserved acidic residues, harboring 2 divalent metal ion binding sites. Structurally similar active sites are also present in Thermus aquaticus Pol I 5′- nuclease domain and in T4 Rnase H.10 Interestingly, when superimposed with the T5 5′-3′ exonuclease, 3 acidic AF0591 residues, D17, D106, and E108, overlap with D26, D153, and D155 of the active site with a RMSD of 0.52 Å (over all atoms, except the Cγ and carboxyl group of E108, for which no electron density was observed). The same AF0591 residues superimpose with D19, D155, and D157 of the T4 Rnase H active site with an RMSD of 0.54 Å. Another acidic AF0591 residue, D88, corresponds approximately to D130 that lies between 2 other acidic residues, E128 and D131, in the T5 5′-3′ exonuclease active site (Fig. 2C). This structural evidence allows us to speculate that the AF0591 catalytic mechanism and reaction may be similar to that of T5 5′-3′ exonuclease or T4 Rnase H. At this stage, it is difficult to specify the biologically relevant oligomerization state of the AF0591. The crystallographic packing in the AF0591 structure shows a dimer is formed through extensive interactions of the ends of the C-terminal β-strand β5 of each subunit, which intertwine into a very long 2-stranded, antiparallel β-sheet (residues 124–132). This dimer interface accounts for a buried surface area of 1174 Å2 per monomer [Fig. 2(A)]. However, this dimerization through the edge β5 strands might be a crystal-packing artifact. In the homologous T5 5′-3′ exonuclease structure, the equivalent β5 strand makes a β-turn in the middle of the corresponding sequence and then continues back as the β6-strand, antiparallel with β5, which suggests that the AF0591 β-strand may have partially unfolded in the crystal to participate in the packing interactions. Thus, further functional studies will be needed to determine the oligomerization state of AF0591. According to fold and function assignment system (FFAS),11 AF0591 has one paralogue in the T. maritima proteome: TM0495, with 23% sequence identity. Models for AF0591 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=2650039. The AF0591 structure reported here represents a new fold as judged by the Structural Classification of Proteins (SCOP) database criteria12 and the first PIN (PilT N-terminus) domain, whose structure has been determined by X-ray crystallography using the MAD method. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the functional determinants of this protein family and the thermostability of these organisms. Protein production and crystallization: AF0591 (TIGR: AF0591; SwissProt:O29664) was amplified by polymerase chain reaction (PCR) from A. fulgidus DSM 4304 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of AF0591. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag consisting of MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in selenomethionine-containing medium using the Escherichia coli methionine auxotrophic strain DL41. Bacteria were lysed by sonication in lysis buffer (50 mM KPO4, pH 7.8, 300 mM NaCl, 10% glycerol, 5mM imidazole, Roche ethylenediaminetetraacetic acid (EDTA)-free protease inhibitor tablets) with 0.5 mg/mL lysozyme. Immediately after sonication, the cell debris was pelleted by ultracentrifugation at 60,000 g for 20 min (4°C). The soluble fraction was applied to a gravity flow metal chelate column (Talon resin charged with cobalt; Clontech) equilibrated in lysis buffer. The column was then washed with 7 column volumes (CV) of wash buffer (20 mM Tris, pH 7.8, 300 mM NaCl, 10% glycerol, 10 mM imidazole) and eluted with 3 CV of elute buffer (25 mM Tris 7.8, 300 mM NaCl, 150 mM imidazole). The protein was then buffer exchanged into 10 mM Tris, pH 7.8, 150 mM NaCl and concentrated to 8 mg/mL by centrifugal ultrafiltration (Orbital). The protein was either frozen in liquid nitrogen for later use or used immediately for crystallization trials. The protein was crystallized using the nanodroplet vapor diffusion method13 with standard JCSG crystallization protocols.5 The crystallization solution contained 30% 2-methyl 1-2,4-pentanediol (MPD) and 0.1 M 2-morpholinoethan sulfonic acid (MES) at pH 6.0. The crystals were indexed in the trigonal space group P3121 (Table I). Data collection: Anomalous diffraction data were collected at Stanford Synchrotron Radiation Laboratory (SSRL; Stanford, CA) on beamline 11-1 at wavelengths corresponding to the inflection point (λ1), peak (λ2), and high-energy remote (λ3) of a selenium MAD experiment, as well as a native 1.9-Å high-resolution data set (λ0), using the BLU-ICE14 data collection environment (Table I). The data sets were collected at 100 K using a Quantum 315 charge-coupled device (CCD) detector. Data were integrated and reduced using MOSFLM15 and then scaled with the program SCALA from the CCP4 suite.16 Data statistics are summarized in Table I. Structure solution and refinement: The structure was determined using the CCP4 suite16 and SOLVE/RESOLVE.17 Structure refinement was performed using REFMAC5,16 O18 and Xfit.19 Refinement statistics are summarized in Table I. The final model includes one protein molecule (residues 10–134) and 115 water molecules in the asymmetric unit. No electron density was observed for residues 1–9, 135, and the expression and purification tags. Figures were prepared with PYMOL (DeLano Scientific). Validation and deposition: Analysis of the stereochemical quality of the models was accomplished using Procheck 3.4 and SFcheck 4.0.7, 16 Comparison of the Asp, Asp, Glu motif from AF0591 with other Asp, Asp, Glu and Asp, Asp, Asp motifs in proteins of known structure was performed with SPASM.20 Atomic coordinates and experimental structure factors of AF0591 have been deposited with the PDB and are accessible under the code 1o4w. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a National user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences).

The TM1478 gene of Thermotoga maritima encodes a methionine aminopeptidase (MAP; EC 3.4.11.18) with a molecular weight of 27,356 Da (250 residues) and a calculated isoelectric point of 6.6. MAP removes the ubiquitous N-terminal methionine from nascent proteins. MAP sequences are conserved from bacteria to humans, and MAP proteins usually contain a dinuclear metal center essential for activity.1 Human MAP is a molecular target for potent antiangiogenesis agents currently in clinical trials.2 Here, we report the crystal structure of TM1478 determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).3 The structure of TM1478 [Fig. 1(A)] was determined to 1.90 Å resolution by molecular replacement (MR) using the related MAP from Escherichia coli with a sequence identity of 42% as a search model [Protein Data Bank (PDB) code: 4MAT].4 Data collection, model, and refinement statistics are summarized in Table I. The final model includes 1 protein monomer (residues 6–254) and 195 water molecules. The Matthews coefficient (Vm) for TM1478 is 2.6 Å3/Da, and the estimated solvent content is 52.6%. The Ramachandran plot, produced by PROCHECK 3.4,5 shows that 95% of the residues are in the most favored regions, and 4.5% are in additional allowed regions. One residue, Asn77 (ϕ = 50.6°, ψ = 110.7°), is in a disallowed region. This residue is characteristic of all MAPs and is the second residue contained within a distorted type II′ β-turn (residues 76–79), observed in all structures thus far studied. The ϕ and ψ angles of Asn77 slightly deviate from the canonical values of a type II′ β turn (ϕ = 60.0°, ψ = −120.0°) by 9.4° and 10.7°, respectively. This distortion of geometry from the ϵ region of the Ramachandran plot is most likely induced by a putative sodium ion coordinated to the carboxyl oxygens of Asn77, Cys234, and Glu79. Although this ion has been observed in other MAP structures (PDB code: 2mat, 3mat, 1c21),4 it is not always assigned to be a metal, and the equivalent position has been attributed to be a water molecule (PDB code: 1mat). One cis-proline is also observed in the structure at position 184. Crystal structure of TM1478. (A) Ribbon diagram of Thermotoga maritima TM1478 color coded from N-terminus (blue) to C-terminus (red) showing the domain organization and the location of the active site (arrow). Helices (H1–H6) and β-strands (β1–β14) are indicated. (B) Diagram showing the secondary structure elements in TM1478 superimposed on its primary sequence with β-hairpins depicted in red and β-sheets labeled in red as A–D. The final model of the TM1478 monomer consists of a single polypeptide chain of 249 amino acids composed of 6 helices (H1–H6) and 14 β-strands (β1–β14). The total α-helix, 310-helix, and β-strand content is 27.3%, 2.4%, and 24.5%, respectively [Fig. 1(A and B)]. TM1478 is composed of two highly similar α/β domains characteristic of the “creatinase/aminopeptidase” fold. Domain 1 comprises residues 6–121 that contains the 3-stranded antiparallel β-sheet A (β2–β4) with 123 topology surrounded by 2 α-helices (H1, H3) and two 310-helices (H2, H4). Domain 2 comprises residues 122–254 and consists of 2 α-helices (H5, H6) followed by 3 separate antiparallel β-sheets (B–D). β-Sheet B comprises 3 β-strands (β5, β9, β12) with 123 topology, β-sheet C comprises 5 β-strands (β6–β8, β13, β14) with 21345 topology, and β-sheet D comprises 2 β-strands (β10, β11) [Fig. 1(A)]. The active site is located in the deep crevice adjacent to β-sheets A and C between the two domains [Fig. 2(A and B)]. Interestingly, no bound metal ions are found in the active site of TM1478 contrary to the expectation for this family of enzymes.4 His171 adopts a dual conformation, one in which the histidine residue resides in the canonical position ready to coordinate a divalent metal ion, whereas the other conformer points away from the active site [Fig. 2(B)]. Although not absolutely necessary for the function of the enzyme,4 it is interesting to speculate that this histidine may act as some sort of switch to stabilize metal binding. (A) Ribbon diagram of a superposition of TM1478 (cyan) and MAP from E. coli (grayish; PDB code: 1MAT). The active site residues are shown in ball and stick. (B) Close-up of the active site using superposition shown in (A). For comparison, the dinuclear cobalt center, its coordinating residues, and a bound methionine, as observed in the MAP structure from E. coli, are shown in ball and stick with carbon atoms colored gray, residue labels shown in brackets, and coordinating bonds to the metal shown in yellow dashes. The corresponding residues in TM1478 are depicted with their carbon atoms colored cyan. A structural similarity search, performed with the coordinates of TM1478 using the DALI server,6 indicates that the closest structural homologue is MAP from E. coli (PDB code: 1MAT).4 The root-mean-square deviation (RMSD) between TM1478 and MAP from E. coli was 1.2 Å over 228 aligned residues with 42% sequence identity [Fig. 2(A)]. According to FFAS,7 TM1478 has only one distant homologue in the T. maritima proteome: TM0042 (aminopeptidase P) with 26% sequence identity. Models for TM1478 homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=tm1478. The crystal structure reported here represents a MAP from T. maritima. TM1478 is the first structure of an apo MAP without any active-site metals. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the functional determinants of this protein family and the thermostability of this enzyme. Protein production and crystallization: TM1478 (TIGR: TM1478; SwissProt: Q9X1I7) was amplified by polymerase chain reaction (PCR) from T. maritima strain MSB8 genomic DNA using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of TM1478. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag consisting of amino acids MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in a modified Terrific Broth [24 g/L yeast extract, 12 g/L tryptone, 1% (v/v) glycerol, 50 mM 3-[N-Morpholino] propanesulfonic acide (MOPS), pH 7.6] using the E. coli methionine auxotrophic strain DL41. Lysozyme was added to the culture at the end of fermentation to a final concentration of 1 mg/mL. Bacteria were lysed by sonication after a freeze-thaw procedure in Lysis Buffer [50 mM Tris, pH 7.9, 50 mM NaCl, 1 mM MgCl2, 0.25 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)], and the cell debris was pelleted by centrifugation at 3400 × g for 60 min. The soluble fraction was applied to a metal chelate affinity resin (Amersham Biosciences) and equilibrated with Equilibration Buffer [50 mM potassium phosphate, pH 7.8, 0.25 mM TCEP, 10% (v/v) glycerol, 300 mM NaCl] containing 20 mM imidazole. The resin was washed with Equilibration Buffer containing 40 mM imidazole, and the protein was eluted with Elution Buffer [20 mM Tris, pH 7.9, 10% (v/v) glycerol, 0.25 mM TCEP, 300 mM imidazole). The nickel affinity eluate was buffer exchanged into crystalization buffer (20 mM Tris, pH 7.9, 150 mM NaCl, 0.25 mM TCEP) and concentrated for crystallization assays to 10 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method8 with standard JCSG crystallization protocols.3 The crystallization solution contained 19% isopropanol, 19% polyethylene glycol (PEG-4000), 0.095 M sodium citrate pH 5.6 and 5% glycerol. The crystals were indexed in the tetragonal space group P43212 (Table I). Data collection: Native diffraction data were collected at the Advanced Light Source (ALS, Berkeley, CA) on beamline 5.0.3. The data set was collected at 100 K using a Quantum 4 charge-coupled device (CCD) detector. Data were integrated, reduced, and scaled using the HKL2000 package9 and the CCP4 suite.10 Data statistics are summarized in Table I. Structure solution and refinement: The structure was determined by MR with AMORE10 using the E. coli (PDB code: 4MAT) MAP coordinates as a search model.4 The MR solution had a correlation coefficient of 0.41 and an initial Rcryst of 47.3%. Structure refinement was performed using the Crystallography & NMR System (CNS)11 and model building with O.12 Refinement statistics are summarized in Table I. The final model includes 1 protein molecule (residues 6–254) and 195 water molecules in the asymmetric unit. No electron density was observed for residue 255 and the N-terminal expression and purification tag. Figures 1A and 2 were produced with PYMOL (DeLano Scientific LLC). Figure 1B was produced with PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum). Validation and deposition: Analysis of the stereochemical quality of the models was accomplished using PROCHECK 3.4 and SFCHECK 4.0.5, 10 Atomic coordinates and experimental structure factors of TM1478 have been deposited with the PDB and are accessible under the code 1o0x. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a National user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). Portions of this research were also carried out at The Advanced Light Source (ALS), Berkeley, CA.

Proteins: Structure, Function, and BioinformaticsVolume 56, Issue 1 p. 167-170 Structure Note Crystal structure of a glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima (TM1621) at 1.60 Å resolution Eugenio Santelli, Eugenio Santelli The Burnham Institute, La Jolla, CaliforniaSearch for more papers by this authorRobert Schwarzenbacher, Robert Schwarzenbacher The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorDaniel McMullan, Daniel McMullan The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorTanya Biorac, Tanya Biorac The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorLinda S. Brinen, Linda S. Brinen The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJaume M. Canaves, Jaume M. Canaves The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorJamison Cambell, Jamison Cambell The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorXiaoping Dai, Xiaoping Dai The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorAshley M. Deacon, Ashley M. Deacon The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMarc-André Elsliger, Marc-André Elsliger The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorSaid Eshagi, Said Eshagi The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRoss Floyd, Ross Floyd The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAdam Godzik, Adam Godzik The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorCarina Grittini, Carina Grittini The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorSlawomir K. Grzechnik, Slawomir K. Grzechnik The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorLukasz Jaroszewski, Lukasz Jaroszewski The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorCathy Karlak, Cathy Karlak The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorHeath E. Klock, Heath E. Klock The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Koesema, Eric Koesema The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJohn S. Kovarik, John S. Kovarik The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAndreas Kreusch, Andreas Kreusch The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPeter Kuhn, Peter Kuhn The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorScott A. Lesley, Scott A. Lesley The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorTimothy M. McPhillips, Timothy M. McPhillips The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMitchell D. Miller, Mitchell D. Miller The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAndrew Morse, Andrew Morse The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorKin Moy, Kin Moy The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJie Ouyang, Jie Ouyang The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorRebecca Page, Rebecca Page The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorKevin Quijano, Kevin Quijano The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorFred Rezezadeh, Fred Rezezadeh The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAlyssa Robb, Alyssa Robb The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Sims, Eric Sims The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorGlen Spraggon, Glen Spraggon The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRaymond C. Stevens, Raymond C. Stevens The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorHenry van den Bedem, Henry van den Bedem The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJeff Velasquez, Jeff Velasquez The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJuli Vincent, Juli Vincent The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorFrank von Delft, Frank von Delft The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorXianhong Wang, Xianhong Wang The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorBill West, Bill West The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorGuenter Wolf, Guenter Wolf The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorQingping Xu, Qingping Xu The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKeith O. Hodgson, Keith O. Hodgson The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJohn Wooley, John Wooley The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, California The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorIan A. Wilson, Corresponding Author Ian A. Wilson wilson@scripps.edu The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaJCSG, Scripps Research Institute, BCC206, 10550 North Torrey Pines Road, La Jolla, CA 92037===Search for more papers by this author Eugenio Santelli, Eugenio Santelli The Burnham Institute, La Jolla, CaliforniaSearch for more papers by this authorRobert Schwarzenbacher, Robert Schwarzenbacher The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorDaniel McMullan, Daniel McMullan The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorTanya Biorac, Tanya Biorac The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorLinda S. Brinen, Linda S. Brinen The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJaume M. Canaves, Jaume M. Canaves The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorJamison Cambell, Jamison Cambell The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorXiaoping Dai, Xiaoping Dai The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorAshley M. Deacon, Ashley M. Deacon The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMarc-André Elsliger, Marc-André Elsliger The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorSaid Eshagi, Said Eshagi The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRoss Floyd, Ross Floyd The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAdam Godzik, Adam Godzik The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorCarina Grittini, Carina Grittini The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorSlawomir K. Grzechnik, Slawomir K. Grzechnik The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorLukasz Jaroszewski, Lukasz Jaroszewski The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorCathy Karlak, Cathy Karlak The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorHeath E. Klock, Heath E. Klock The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Koesema, Eric Koesema The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorJohn S. Kovarik, John S. Kovarik The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAndreas Kreusch, Andreas Kreusch The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorPeter Kuhn, Peter Kuhn The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorScott A. Lesley, Scott A. Lesley The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorTimothy M. McPhillips, Timothy M. McPhillips The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorMitchell D. Miller, Mitchell D. Miller The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAndrew Morse, Andrew Morse The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorKin Moy, Kin Moy The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJie Ouyang, Jie Ouyang The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorRebecca Page, Rebecca Page The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorKevin Quijano, Kevin Quijano The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorFred Rezezadeh, Fred Rezezadeh The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorAlyssa Robb, Alyssa Robb The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorEric Sims, Eric Sims The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorGlen Spraggon, Glen Spraggon The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorRaymond C. Stevens, Raymond C. Stevens The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorHenry van den Bedem, Henry van den Bedem The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJeff Velasquez, Jeff Velasquez The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorJuli Vincent, Juli Vincent The Joint Center for Structural Genomics, La Jolla, California The Genomics Institute of the Novartis Research Foundation, San Diego, CaliforniaSearch for more papers by this authorFrank von Delft, Frank von Delft The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaSearch for more papers by this authorXianhong Wang, Xianhong Wang The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorBill West, Bill West The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, CaliforniaSearch for more papers by this authorGuenter Wolf, Guenter Wolf The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorQingping Xu, Qingping Xu The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorKeith O. Hodgson, Keith O. Hodgson The Joint Center for Structural Genomics, La Jolla, California Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CaliforniaSearch for more papers by this authorJohn Wooley, John Wooley The Joint Center for Structural Genomics, La Jolla, California The San Diego Supercomputer Center, La Jolla, California The University of California, San Diego, La Jolla, CaliforniaSearch for more papers by this authorIan A. Wilson, Corresponding Author Ian A. Wilson wilson@scripps.edu The Joint Center for Structural Genomics, La Jolla, California The Scripps Research Institute, La Jolla, CaliforniaJCSG, Scripps Research Institute, BCC206, 10550 North Torrey Pines Road, La Jolla, CA 92037===Search for more papers by this author First published: 07 May 2004 https://doi.org/10.1002/prot.20120Citations: 25Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume56, Issue11 July 2004Pages 167-170 RelatedInformation

In order to extend the structural coverage of eukaryotic genomes, we selected 288 open reading frames (ORF's) in the yeast genome with significant homology to mouse proteins. One of these, an allantoicase (YIR029W) from Saccharomyces cerevisiae, encodes a protein with a molecular weight of 38,581 Da (residues 1–343) and a calculated isoelectric point of 5.9. Allantoicase (EC 3.5.3.4), also known as allantoate amidinohydrolase, is involved in purine degradation and facilitates the utilization of purines as secondary nitrogen sources in nitrogen-limiting conditions.1 While purine degradation converges to uric acid in all vertebrates, its further degradation varies from species to species. Uric acid is excreted by birds, reptiles, and some mammals that do not have a functional uricase gene, whereas other mammals produce allantoin. Amphibians and microorganisms produce ammonia and carbon dioxide using the uricolytic pathway.2 Allantoicase performs the second step in this pathway that hydrolyses the linear amidine allantoate to (−)-ureidoglycolate and urea. Hydrolysis of the alternative substrate (+)-ureidoglycolate to glyoxylate and urea has also been observed.3 Although allantoicase activity is not detectable in mammals, birds, reptiles and some fishes, they still contain the gene for allantoicase, suggesting an alternative function.2, 4 Here, we report the crystal structure of YIR029W determined using the semiautomated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).5 The structure of YIR029W [Fig. 1(A)] was determined to 2.40-Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes one protein monomer (residues 1–57, 67–187, 194–284 and 292–343) and 239 water molecules. No electron density was observed for residues 58–66, 188–193 and 285–295. The Matthews' coefficient (Vm)6 for YIR029W is 2.87 Å3/Da and the estimated solvent content is 56.8%. The Ramachandran plot, produced by Procheck 3.47 shows that 85.5% of the residues are in the most favored regions and 14.5% are in additional allowed regions. Crystal structure of YIR029W. A: Stereo ribbon diagram of Saccharomyces cerevisiae YIR029W color coded from N-terminus (blue) to C-terminus (red) showing the domain organization. Helices H1–H7, and β-strands (β1–β24) in β-sheets A–F are indicated. Residue numbers at the beginning and end of missing loops are indicated. B: Diagram showing the secondary structure elements in YIR029W superimposed on its primary sequence. The disordered regions are depicted by a dashed line with the corresponding sequence in brackets. The β-sheet designation of the individual β-strands are indicated by a red A–F and β-hairpins are depicted as red loops. The YIR029W monomer contains 24 β-strands (β1–β24), one α-helix (H2) and six 310–helices (H1, H3–H7) [Fig. 1(A, B)]. The total β-strand, α-helical and 310-helical content is 40.9%, 2.5% and 6.6% respectively. YIR029W contains an N-terminal strand-helix motif and two allantoicase-repeats (AR) which form two similar β-sandwich domains.8 AR1 (residues 21–187) and AR2 (residues 194–351) are connected by a flexible linker (residues 188–193) [Fig. 1(A)]. AR1 folds in a β–sandwich composed of four-stranded (A) and five-stranded (B) antiparallel β-sheets: A with 1423 topology (β2, β6, β9, β12) and B with 12534 topology (β3, β5, β7, β8, β10). The A and B β-sheets are slightly crossed (∼40°) with respect to each other and bury a compact hydrophobic core. The β-strands are connected by extended loops one of which is disordered (residues 58–66) in the crystal structure. In addition, a short, additional two-stranded, antiparallel β-sheet C (β4, β11) is flanked by two loops and helix H4. The N-terminal strand-helix motif (β1, H1, H2; residues 1–20) packs against β-sheet A, where β1 forms an additional, antiparallel β-strand that is hydrogen bonded to β9. H2 forms part of the interface to AR2 through interaction with β14. AR2 has a very similar fold to AR1. Both domains are related by an approximate two-fold and can be superimposed with a root-mean-square deviation (RMSD) of 1.04 Å for 115 residues with 40% sequence identity. AR2 folds into a β–sandwich composed of a four-stranded (D) and a five-stranded (E) antiparallel β-sheet: D with 1423 topology (β13, β18, β21, β24) and E with 12534 topology (β14, β17, β19, β20, β22) [Fig. 1(A, B)]. The β-strands are connected by extended loops one of which is disordered (residues 285–295). In addition, a short, additional three-stranded antiparallel β-sheet F (β15, β16, β23) is flanked by two loops and helix H6 next to the interface region with AR1. A structural similarity search, performed with the coordinates of YIR029W using the DALI server,9 showed the best match to be the N-terminal domain of the human DNA-Repair Protein XRCC1 (PDB:1xna),10 with an RMSD of 2.5 Å over 123 aligned residues with 17% sequence identity to the AR2 domain. XRCC1 is also similar to the AR1 domain, where the RMSD is 2.6 Å over 122 aligned residues with 16% sequence identity. Another structural homologue is the galactose-binding domain in a sialidase from M. viridifaciens (PDB:1euu),9 where the respective RMSD's for the AR1 and AR2 domains are 2.8 Å and 2.7 Å over 118 aligned residues with 10% and 11% sequence identity. None of the DALI hits contains a second AR domain, indicating that YIR029W is the first structure of a protein containing two Allantoicase repeats. Models for YIR029W homologues can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=YIR029W. The crystallographic packing in the YIR029W structure indicates that a hexamer is the biologically-relevant oligomeric form. A hexamer (200 kDa), composed of two trimers (100 kDa), has also been reported in biophysical studies with the allantoicase from Chlamydomonas reinhardtii.1 The hexamer is comprised of two planar trimers stacked on top of each other to form a barrel-like structure with 3,2-symmetry. The hexamer measures 100 Å in diameter and 60 Å in height with a 15 Å wide inner channel [Fig. 2(A)]. The interfaces in the trimer are formed by head-to-tail interactions between residues from the AR1 domain (Glu72, Arg75, Glu78, and Asp172) from one subunit with the AR2 domain (Arg238, Arg240, Lys305, and Asp332) from the adjacent subunit [Fig. 2(A)]. The interface between the two trimers is formed by side-on interactions between residues from the AR1 domain (strand β3 and the loop-region of residues 122–127) from one subunit with residues from the AR2 domain (loop-region of residues 194–203 and β-sheet D) from the other subunit. The subunit interactions are stabilized by seven salt-bridges and account for a buried surface area of 2444 Å2 per monomer. A: The YIR029W hexamer in surface representation, shown normal and parallel to the three-fold. Upper and lower trimers are colored grey and green, respectively. One subunit of the gray trimers is shown in blue and the clusters of conserved residues are highlighted in yellow. B: Ribbon diagram of a superposition of YIR029W AR1 (residues 1–187) in yellow and AR2 (residues 193–343) in blue. The cluster of conserved residues in AR1 and AR2 (labels shown in brackets) is shown in ball and stick. C: Ribbon diagram of a superposition of YIR029W AR2 (residues 193–343) in blue and the galactose-binding domain (PDB:1euu) in white with bound substrate (D-galactose) The cluster of conserved residues in AR2 (blue) and the D-galactose (white) are shown in ball and stick. Mapping the sequence conservation of 43 known allantoicases8 onto the YIR029W structure identifies two highly similar clusters of hydrophilic residues in AR1 (Glu72, Arg75, Asp82, Asn108, and Asp172) and in AR2 (Glu235, Arg238, Asp246, Asn272, and Asp332) [Fig. 2(A,B)]. The two conserved clusters are within the head-to-tail subunit interface [Fig. 2(A)]. The active site in the galactose-binding domain coincides with the conserved cluster in the AR-repeats of YIR029W, suggesting a possible location of its active site in the subunit interface [Fig. 2(C)]. An alternative active site location is the deep crevice in the AR1-AR2 domain interface [Fig. 2(A)], which contains two strictly conserved residues (Asp24 and Arg179). The YIR029W structure reported here represents the first allantoicase, whose structure has been determined by X-ray crystallography using the MAD method. The information reported here, in combination with further biochemical and biophysical studies will yield valuable insights into the functional role of allantoicase in microorganisms, invertebrates, and vertebrates. YIR029W (TIGR: YIR029W; Swissprot:O29664) was amplified by PCR from genomic DNA from Saccharomyces cerevisiae using Taq T33 polymerase (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of YIR029W. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag consisting of MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in selenomethionine-containing medium using the E. coli methionine auxotrophic strain DL41. Bacteria were lysed by sonication in lysis buffer (50 mM KPO4, pH 7.8, 300 mM NaCl, 10% glycerol, 5 mM imidazole, Roche EDTA-free protease inhibitor tablets) with 0.5 mg/ml lysozyme. Immediately after sonication, the cell debris was pelleted by ultracentrifugation at 60,000 g for 20 min (4°C). The soluble fraction was applied to a gravity flow metal chelate column (Talon resin charged with cobalt; Clontech) equilibrated in lysis buffer. The column was then washed with seven column volumes (CV) of wash buffer (20 mM Tris, pH 7.8, 300 mM NaCl, 10% glycerol, 10 mM imidazole) and eluted with 3 CV of elute buffer (25 mM Tris, pH 7.8, 300 mM NaCl, 150 mM imidazole). The protein was then buffer exchanged into crystallization buffer (10 mM Tris, pH 7.8, 150 mM NaCl) and concentrated to 8 mg/mL by centrifugal ultrafiltration (Orbital). The protein was either frozen in liquid nitrogen for later use or used immediately for crystallization trials. The protein was crystallized using the nanodroplet vapor diffusion method12 with standard Joint Center for Structural Genomics crystallization protocols.5 Crystals grew in 30% ethylene glycol. The crystals were indexed in the hexagonal space group P6322 (Table I). Anomalous diffraction data were collected at the Stanford Synchrotron Radiation Laboratory (SSRL, Stanford, CA) on beamline 11-1 at wavelengths corresponding to the inflection point (λ1), peak (λ2), and high energy remote (λ3) of a selenium MAD experiment, in addition to a 2.40 Å native high resolution data set (λ0), using the BLU-ICE13 data collection environment (Table I). The data sets were collected at 100 K using a Quantum 315 CCD detector. Data were integrated and reduced using Mosflm14 and then scaled with the program SCALA from the CCP4 suite.15 Data statistics are summarized in Table I. The structure was determined using the CCP4 suite15 and SOLVE/RESOLVE.16 Structure refinement was performed using REFMAC5,15 O,17 and Xfit.18 Refinement statistics are summarized in Table I. The final model includes one protein monomer (residues 1–57, 67–187, 194–284, and 292–343), one histidine residue from the purification tag, and 239 water molecules in the asymmetric unit. No electron density was observed for residues 58–66, 188–193, and 285–291 and the rest of the expression and purification tag. Analysis of the stereochemical quality of the model was accomplished using Procheck 3.4,7 SFcheck 4.0,15 and WHAT IF 5.0.19 Protein quarternary structure analysis used the PQS server (http://pqs.ebi.ac.uk/). Figure 1(B) was adapted from an analysis using PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/) and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of YIR029W have been deposited with the Protein Data Bank and are accessible under the code 1o59. This work was supported by NIH Protein Structure Initiative grant P50-GM 62411 from the National Institute of General Medical Sciences (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences).

In order to extend the structural coverage of eukaryotic genomes, we selected 288 open reading frames (ORF's) in the yeast genome with significant homology to mouse proteins. One of these, a hydrolase (YDR428C) from Saccharomyces cerevisiae, encodes a protein with a molecular weight of 29,859 Da (residues 1–261) and a calculated isoelectric point of 5.2. The ESTHER database of hydrolase enzymes (http://bioweb.ensam.inra.fr/esther)1 classifies it as a serine hydrolase belonging to the subfamily of hormone-sensitive lipase-like hydrolases, which has over 250 homologs in all kingdoms of life. Mutations in genes of this superfamily are associated with diseases such as autism, goiter, increased risk factor of late onset Alzheimer disease, and xenobiotic sensitivities, including hypersensitivity to acetylcholinesterase inhibitors.1 Here, we report the crystal structure of this putative α/β-serine hydrolase determined using the semi-automated high-throughput pipeline of the Joint Center for Structural Genomics (JCSG).2 The structure of YDR428C [Fig. 1(A)] was determined to 1.85 Å resolution using the multi-wavelength anomalous dispersion (MAD) method. Data collection, model, and refinement statistics are summarized in Table I. The final model includes two protein monomers (residues 4–261 molecule A and residues 10–261 molecule B), two glycerol molecules, one chloride ion, and 454 water molecules. No electron density was observed for residues 2–3 in chain A and residues 1–9 in chain B. The Matthews' coefficient (Vm)3 for YDR428C is 2.10 Å3/Da and the estimated solvent content is 41.2%. The Ramachandran plot, produced by MolProbity,4 shows that 98% of the residues are in the favored regions and 2% are in additional allowed regions. Crystal structure of YDR428C. A: Stereoview of ribbon diagram of Saccharomyces cerevisiae YDR428C color coded from N-terminus (blue) to C-terminus (red) showing the domain organization. α-Helices H1–H12, and β-strands (β1–β7) are indicated. B: Diagram showing the secondary structure elements in YDR428C superimposed on its primary sequence. The α-helices, 310-helices, β-sheet strands (red A), β-bulges, and γ-turns are indicated. The disordered regions are depicted by a dashed line with the corresponding sequence in brackets. The final model of the YDR428C monomer consists of seven β-strands (β1–β7), with 1324567 topology, twelve α-helices (H1–H12) and four 310-helical segments (H2′, H4′, H7′, H9′) [Fig. 1(A,B)]. The total β-strand, α-helical, and 310-helical content is 16.1%, 53.3%, and 1.9%, respectively. YDR428C contains an α/β hydrolase fold5, 6 with a central, seven-stranded β-sheet, comprised of six parallel β-strands (β2–β7) that is preceded by an additional anti-parallel, N-terminal strand β1 [Fig. 1(A)]. The β-sheet is twisted and flanked by nine α-helices (H1–H5, H9–H12) [Fig. 1(A)]. An additional lid subdomain of three α-helices (H6–H8; residues 155–181) is inserted after the β5 strand [Fig. 1(A,B)]. Similar lid subdomains in several lipase structures have been shown to undergo a conformational change upon substrate binding.7 Further functional and structural studies of YDR428C will be needed to determine if this occurs with this lid subdomain. The α/β-hydrolase fold is usually similar to a scaffold for a catalytic triad of three residues: a nucleophile, His, and an acid residue. As for other α/β hydrolases, YDR428C contains a signature motif of 108-GXNuXG-112 (where Nu = nucleophile) with S110 as its "nucleophile elbow" with energetically-strained, main-chain torsional angles (ϕ = 63°, ψ = −134° for one subunit and ϕ = 54°, ψ = −130° for the other subunit) [Fig. 2(B)].5, 6 The three-dimensional structure of YDR428C reveals that the other two residues of the catalytic triad are composed of D211 and H243 [Fig. 2(A,B)], and could not have been predicted from sequence information alone due to a lack of consensus sequence. These active site residues are located at the bottom of a deep cleft. The residues that line this cleft are entirely hydrophobic (A39, W40, V111, I153, L156, L159, Y166, F169, L213, L214) and are likely to be important for substrate discrimination. A: Ribbon diagram of a superposition of YDR428C (blue) and P. fluorescens carboxylesterase (grey). Residues of the catalytic triad, shown in ball and stick, indicate the active site location. B: Same as A, but a close up view of the catalytic triad. Active site residues as observed in P. fluorescens carboxylesterase (PDB 1auo; grey; P. fluorescens residues shown in parenthesis) and their counterparts in YDR428C (blue) are shown in ball and stick. Hydrogen bond interactions and distances are indicated. C: Ribbon diagram of the YDR428C dimer. β7 strands and helices H10 and H12 forming part of the dimer interface and locations of active sites (arrow) are indicated. A structural similarity search, performed with the coordinates of YDR428C using the DALI server,8 indicates structural similarity to many other α/β hydrolases, but particularly to carboxylesterase from Pseudomonas fluorescens (PDB: 1auo).9 The RMSD is 2.5 Å over 184 aligned residues with 14% sequence identity [Fig. 2(A)]. Another close structural homolog is brefeldin A esterase from Bacillus subtilis (PDB: 1jkm),10 where the RMSD is 2.7 Å over 220 aligned residues with 11% sequence identity. Models for YDR428C homologs can be accessed at http://www1.jcsg.org/cgi-bin/models/get_mor.pl?key=YDR428C. For P. fluorescens carboxylesterase, the active site contains a catalytic triad of residues S114, D168, and H199.9 Structurally similar active sites are also present in B. subtilis brefeldin A esterase.10 Interestingly, when superimposed with the P. fluorescens carboxylesterase, three YDR428C residues, S110, D211, and H243, closely overlap with S114, D168, and H199 of the active site with an RMSD of 0.18 Å (all atoms) [Fig. 2(B)]. The same YDR428C residues superimpose with S202, D308, and H338 of the B. subtilis brefeldin A esterase active site with an RMSD of 0.25 Å. The arrangement of the YDR428C catalytic triad is consistent with those of other catalytic triads found in other lipases and esterases.5, 6 Thus, the YDR428C catalytic mechanism and reaction may be similar to those of carboxylesterase or brefeldin A esterase. The noncrystallographic symmetry in the YDR428C crystal results in dimer formation through extensive association of the C-terminal β-strands β7 (residues F233, K234, L235, Y236, L237) of each subunit in an antiparallel fashion leading to a formation of a fourteen-stranded, highly curved β-sheet [Fig. 2(C)]. Additional interactions are formed by the C-terminal α-helices H10 (residues L216, N220, I223, S224, Q227) and H12 (residues D258, N259, C261) from each subunit. This extensive dimer interface accounts for a buried surface area of 1803 Å2 for each monomer.11 The two subunits are believed to act independently because of the distance between their respective active sites (∼36 Å). In support of YDR428C being a crystallographic dimer, the homologous B. subtilis brefeldin A esterase is also a homodimer in solution.10 Interestingly, the structure to YDR428C with equivalent secondary structures forming the dimer interface with the exception being that one of the subunits of brefeldin A esterase is shifted in the plane of the C-terminal β-strands relative to that in the YDR428C dimer. Despite the similarity to brefeldin A esterase, the YDR428C dimerization might still be due to crystal packing. Thus, further functional studies will be needed to confirm its oligomerization state. The YDR428C structure reported here represents a putative hormone-sensitive lipase from yeast, whose structure has been determined by X-ray crystallography using the MAD method. The information reported here, in combination with further biochemical and biophysical studies, will yield valuable insights into the functional determinants of the equivalent protein in mammals. YDR428C (TIGR: YDR428C; Swissprot: S69709) was amplified by PCR from genomic DNA from Saccharomyces cerevisiae using PfuTurbo (Stratagene) and primer pairs encoding the predicted 5′- and 3′-ends of YDR428C. The PCR product was cloned into plasmid pMH1, which encodes an expression and purification tag consisting of MGSDKIHHHHHH at the amino terminus of the full-length protein. The cloning junctions were confirmed by sequencing. Protein expression was performed in selenomethionine-containing medium using the E. coli methionine auxotrophic strain DL41. Bacteria were lysed by sonication in lysis buffer (50mM K2HPO4, pH 7.8, 300 mM NaCl, 10% glycerol, 5 mM imidazole, Roche EDTA-free protease inhibitor tablets) with 0.5 mg/ml lysozyme. Immediately after sonication, the cell debris was pelleted by ultracentrifugation at 60,000 g for 20 min (4°C). The soluble fraction was applied to a gravity flow metal chelate column (Talon resin charged with cobalt; Clontech) equilibrated in lysis buffer. The column was then washed with 7 column- volumes (CV) of wash buffer (20 mM Tris, pH 7.8, 300 mM NaCl, 10% glycerol, 10 mM imidazole) and eluted with 3 CV of elute buffer (25 mM, Tris 7.8, 300 mM NaCl, 150 mM imidazole). The protein was then buffer exchanged into 10 mM Tris (pH 7.8), 150 mM NaCl and concentrated to 13 mg/mL by centrifugal ultrafiltration (Orbital). The protein was either frozen in liquid nitrogen for later use or used immediately for crystallization trials. The protein was crystallized using the nanodroplet vapor diffusion method12 with standard JCSG crystallization protocols.2 The crystallization solution contained 17% PEG MME 2000, 10% glycerol, and 100 mM HEPES at pH 7.0. Thecrystals were indexed in the monoclinic space group P21 (Table I). Anomalous diffraction data were collected at the Advanced Photon Source (APS, Argonne, USA) on beamline NE-CAT-8BM at wavelengths corresponding to the inflection point (λ1), high energy remote (λ2), and peak (λ3) of a selenium MAD experiment using the ADSC data collection environment (Table I). The data sets were collected at 100 K using a Quantum 315 CCD detector. Data were integrated, reduced, and scaled using HKL2000.13 Data statistics are summarized in Table I. The structure was determined by using the CCP4 suite14 and SOLVE/RESOLVE.15 Structure refinement was performed using REFMAC5,14 O,16 and Xfit.17 Refinement statistics are summarized in Table I. The final model includes two protein monomers, residues 4–261 for molecule A, and two histidine residues (−1 and 0) of the purification tag, and residues 10–261 for molecule B, 454 water molecules, two glycerol molecules, and a chloride ion. No electron density was observed for residues 2–3 and the first 10 residues of N-terminal expression and purification tag of molecule A, as well as residues 1–9 and the entire tag of molecule B. Analysis of the stereochemical quality of the model was accomplished using the AutoDepInputTool (http://deposit.pdb.org/adit/), MolProbity,4 SFcheck 4.0,18 and WHAT IF 5.0.19 Protein quaternary structure analysis used GRASP.11 Figure 1(B) was adapted from an analysis using PDBsum (http://www.biochem.ucl.ac.uk/bsm/pdbsum/) and all others were prepared with PYMOL (DeLano Scientific). Atomic coordinates and experimental structure factors of YDR428C have been deposited with the PDB and are accessible under the code 1vkh. This work was supported by NIH Protein Structure Initiative grant P50-GM 62411 from the National Institute of General Medical Sciences (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the National Institutes of Health (National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences). Data collection was conducted at the Northeastern Collaborative Access Team beamlines of the Advanced Photon Source, supported by award RR-15301 from the National Center for Research Resources at the National Institute of Health. Use of the Advanced Photon Source is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under contract No. W-31-109-ENG-38.